High-transduction-efficiency raav vectors, compositions, and methods of use

ABSTRACT

The present invention provides AAV capsid proteins comprising modification of one or a combination of the surface-exposed lysine, serine, threonine and/or tyrosine residues in the VP3 region. Also provided are rAAV virions comprising the AAV capsid proteins of the present invention, as well as nucleic acid molecules and rAAV vectors encoding the AAV capsid proteins of the present invention. Advantageously, the rAAV vectors and virions of the present invention have improved efficiency in transduction of a variety of cells, tissues and organs of interest, when compared to wild-type rAAV vectors and virions.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the priority benefit of U.S. patentapplication Ser. No. 13/840,224, filed Mar. 15, 2013 (pending), and U.S.Provisional Patent Appl. No. 61/647,318, filed May 15, 2012 (pending).The present application is also related to U.S. patent application Ser.No. 12/595,196, filed Dec. 31, 2009 (to issue May 21, 2013 as U.S. Pat.No. 8,445,267), Intl. Patent Appl. No. PCT/US2008/059647 filed Apr. 8,2008 (nationalized), U.S. Provisional Patent Appl. No. 60/910,798, filedApr. 9, 2007 (expired), U.S. patent application Ser. No. 13/854,011,filed Mar. 29, 2013 (pending); and U.S. patent application Ser. No.13/855,610, filed Apr. 2, 2013 (pending). The content of each of theaforementioned applications is hereby incorporated in its entirety byexpress reference thereto.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable.

BACKGROUND OF THE INVENTION Field of the Invention

Major advances in the field of gene therapy have been achieved by usingviruses to deliver therapeutic genetic material. The adeno-associatedvirus (AAV) has attracted considerable attention as a highly effectiveviral vector for gene therapy due to its low immunogenicity and abilityto effectively transduce non-dividing cells. AAV has been shown toinfect a variety of cell and tissue types, and significant progress hasbeen made over the last decade to adapt this viral system for use inhuman gene therapy.

In its normal “wild type” form, recombinant AAV (rAAV) DNA is packagedinto the viral capsid as a single stranded molecule about 4600nucleotides (nt) in length. Following infection of the cell by thevirus, the molecular machinery of the cell converts the single DNAstrand into a double-stranded form.

AAV has many properties that favor its use as a gene deliveryvehicle: 1) the wild type virus is not associated with any pathologichuman condition; 2) the recombinant form does not contain native viralcoding sequences; and 3) persistent transgenic expression has beenobserved in many applications. One of the main obstacles of the genetherapy, the induction of immuno-competition in cellular immuneresponses against vector-derived and transgene-derived epitopes, can beovercome by replication-deficiency and lack of viral proteins expressedby recombinant AAV.

The transduction efficiency of recombinant adeno-associated virusvectors varies greatly in different cells and tissues in vitro and invivo. Systematic studies have been performed to elucidate thefundamental steps in the life cycle of AAV. For example, it has beendocumented that a cellular protein, FKBP52, phosphorylated at tyrosineresidues by epidermal growth factor receptor protein tyrosine kinase(EGFR-PTK), inhibits AAV second-strand DNA synthesis and consequently,transgene expression in vitro as well as in vivo. It has also beendemonstrated that EGFR-PTK signaling modulates the ubiquitin/proteasomepathway-mediated intracellular trafficking as well as FKBP52-mediatedsecond-strand DNA synthesis of AAV vectors. In those studies, inhibitionof EGFR-PTK signaling led to decreased ubiquitination of AAV capsidproteins, which in turn, facilitated nuclear transport by limitingproteasome-mediated degradation of AAV vectors, implicatingEGFR-PTK-mediated phosphorylation of tyrosine residues on AAV capsids.

BRIEF SUMMARY OF THE INVENTION

The present invention provides AAV capsid proteins comprisingmodification of one or a combination of the surface-exposed lysine,serine, threonine and/or tyrosine residues in the VP3 region. Alsoprovided are rAAV virions that comprise the AAV capsid proteins of thepresent invention, as well as nucleic acid molecules and rAAV vectorsencoding the AAV capsid proteins of the present invention.Advantageously, the rAAV vectors and virions of the present inventionhave improved efficiency in transduction of a variety of cells, tissuesand organs of interest, when compared to wild-type rAAV vectors andvirions.

In one embodiment, the present invention provides a nucleic acidmolecule comprising a nucleotide sequence encoding an AAV capsidprotein, wherein the VP3 region of the AAV capsid protein comprises anon-lysine residue at a position that corresponds to a lysine residue inthe VP3 region of the capsid protein of the wild-type AAV [e.g., SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10; and in oneembodiment, preferably the capsid protein of wild-type AAV2 (SEQ IDNO:2)], wherein the lysine residue in the VP3 region of the wild-typeAAV protein is one or more of K258, K321, K459, K490, K507, K527, K572,K532, K544, K549, K556, K649, K655, K665, and K706, or any combinationthereof.

In one embodiment, the surface-exposed lysine residue corresponding K532of the wild-type AAV2 capsid sequence is modified. In one embodiment,the surface-exposed lysine residue of the AAV capsid is modified intoglutamic acid (E) or arginine (R). In specific embodiments, thesurface-exposed lysine residue corresponding K532 of the wild-type AAV2capsid sequence is modified into arginine (K532R).

In certain embodiments, one or more surface-exposed lysine residuescorresponding to K490, K544, K549, and K556 of the wild-type AAV2 capsidsequence are modified. In certain specific embodiments, one or moresurface-exposed lysine residue corresponding K490, K544, K549, and K556of the wild-type AAV2 capsid sequence are modified into glutamic acid(E).

In one embodiment, the present invention provides AAV2 vectors whereinsurface-exposed lysine residues corresponding to K544 and K556 residuesof the wild-type AAV2 capsid are modified into glutamic acid (E).

In certain embodiments, one or more surface-exposed lysine residuescorresponding to K530, K547, and K569 of the wild-type AAV8 capsidsequence are modified. In certain specific embodiments, one or moresurface-exposed lysine residue corresponding K530, K547, and K569 of thewild-type AAV2 capsid sequence are modified into glutamic acid (E).

In one embodiment, a combination of surface-exposed lysine, serine,threonine and/or tyrosine residues of the AAV capsid is modified,wherein the modification occurs at positions corresponding to

(Y444F+Y500F+Y730F+T491V)

(Y444F+Y500F+Y730F+T491V+T550V)

(Y444F+Y500F+Y730F+T491V+T659V)

(T491V+T550V+T659V)

(Y440F+Y500F+Y730F)

(Y444F+Y500F+Y730F+T491V+S662V), and/or

(Y444F+Y500F+Y730F+T491V+T550V+T659V)

of the wild-type AAV capsid sequence [e.g., one or more of SEQ IDNOs:1-10; and in a particular embodiment, of the capsid protein ofwild-type AAV2 sequence (SEQ ID NO:2)].

In related embodiments, the invention provides methods for using thevectors and compositions disclosed herein, and further providesprocesses for the transduction of one or more cells, one or moretissues, and/or one or more organs of interest, and particularly thoseof a mammalian animal using the disclosed viral vector constructs. In anoverall and general sense, such methods generally include at least thestep of introducing into a suitable host cell of interest, at least afirst composition that comprises, consists essentially of, oralternatively consists of, an effective amount of a rAAV vector and/oran infectious AAV viral particle, or recombinant AAV virion of presentinvention in an amount and for a time sufficient to transform at least afirst cell or a first population of cells with the viral vector. Inparticular embodiments, the vectors, virions, or infectious viralparticles of the present invention are preferably useful as vectors forintroducing one or more nucleic acid segments to a selected host cell ofinterest. Preferably the host cell is a mammalian host cell, with humanhost cells being particularly preferred as targets for the recombinantvectors and virions described herein. In certain embodiments, suchvectors will further comprise one or more isolated DNA segments encodinga selected therapeutic and/or diagnostic agent, including, for exampleone or more polynucleotides comprising one or more genes of interestthat are capable of being expressed in a mammalian host cell that hasbeen transformed by one or more of the vectors, viruses, or infectiousvirions described and provided herein.

In one aspect, the invention further provides compositions comprisingrecombinant adeno-associated viral (AAV) vectors, virions, viralparticles, and pharmaceutical formulations thereof, useful in methodsfor delivering genetic material encoding one or more beneficial ortherapeutic product(s) to mammalian cells and tissues. In particular,the compositions and methods of the invention provide a significantadvancement in the art through their use in the treatment, prevention,and/or amelioration of symptoms of one or more mammalian diseases. It iscontemplated that human gene therapy will particularly benefit from thepresent teachings by providing new and improved viral vector constructsfor use in the treatment of a number of diverse diseases, disorders, anddysfunctions.

In another aspect, the invention concerns modified rAAV vector thatencode one or more mammalian therapeutic agents for the prevention,treatment, and/or amelioration of one or more disorders in the mammalinto which the vector construct is delivered. In particular, theinvention provides rAAV-based expression constructs that encode one ormore mammalian therapeutic agent(s) (including, but not limited to, forexample, protein(s), polypeptide(s), peptide(s), enzyme(s), antibodies,antigen binding fragments, as well as variants, and/or active fragmentsthereof, for use in the treatment, prophylaxis, and/or amelioration ofone or more symptoms of a mammalian disease, dysfunction, injury, and/ordisorder.

In another embodiment, the invention concerns genetically modified rAAVvectors that comprise at least a first nucleic acid segment that encodesone or more therapeutic agents that alter, inhibit, reduce, prevent,eliminate, or impair the activity of one or more endogenous biologicalprocesses in the cell. In particular embodiments, such therapeuticagents may be those that selectively inhibit or reduce the effects ofone or more metabolic processes, dysfunctions, disorders, or diseases.In certain embodiments, the defect may be caused by injury or trauma tothe mammal for which treatment is desired. In other embodiments, thedefect may be caused the over-expression of an endogenous biologicalcompound, while in other embodiments still; the defect may be caused bythe under-expression or even lack of one or more endogenous biologicalcompounds.

When the use of such vectors is contemplated for introduction of one ormore exogenous proteins, polypeptides, peptides, ribozymes, siRNAs,and/or antisense oligonucleotides, to a particular cell transfected withthe vector, one may employ the modified AAV vectors disclosed herein byincorporating into the vector at least a first exogenous polynucleotideoperably positioned downstream and under the control of at least a firstheterologous promoter that expresses the polynucleotide in a cellcomprising the vector to produce the encoded therapeutic agent,including for example, peptides, proteins, polypeptides, antibodies,ribozymes, siRNAs, and antisense oligo- or polynucleotides. Suchconstructs may employ one or more heterologous promoters to express thetherapeutic agent of interest. Such promoters may be constitutive,inducible, or even cell- or tissue-specific. Exemplary promotersinclude, but are not limited to, a CMV promoter, a β-actin promoter, ahybrid CMV promoter, a hybrid β-actin promoter, an EF1 promoter, a U1apromoter, a U1b promoter, a Tet-inducible promoter, a VP16-LexApromoter, a joint-specific promoter and a human-specific promoter.

The genetically-modified rAAV vectors or expression systems of theinvention may also further comprise a second nucleic acid segment thatcomprises, consists essentially of, or consists of, one or moreenhancers, regulatory elements, transcriptional elements, to alter oreffect transcription of the heterologous gene cloned in the rAAVvectors. For example, the rAAV vectors of the present invention mayfurther comprise a second nucleic acid segment that comprises, consistsessentially of, or consists of, at least a first CMV enhancer, asynthetic enhancer, or a cell- or tissue-specific enhancer. The secondnucleic acid segment may also further comprise, consist essentially of,or consist of one or more intron sequences, post-transcriptionalregulatory elements, or such like. The vectors and expression systems ofthe invention may also optionally further comprise a third nucleic acidsegment that comprises, consists essentially of or consists of, one ormore polylinker or multiple restriction sites/cloning region(s) tofacilitate insertion of one or more selected genetic elements,polynucleotides, and the like into the rAAV vectors at a convenientrestriction site.

In aspects of the invention, the exogenous polynucleotides that arecomprised within one or more of the improved rAAV vectors disclosedherein are preferably of mammalian origin, with polynucleotides encodingpolypeptides and peptides of human, primate, murine, porcine, bovine,ovine, feline, canine, equine, epine, caprine, or lupine origin beingparticularly preferred.

As described above, the exogenous polynucleotide will preferably encodeone or more proteins, polypeptides, peptides, enzymes, antibodies,siRNAs, ribozymes, or antisense polynucleotides, oligonucleotides, PNAmolecules, or a combination of two or more of these therapeutic agents.In fact, the exogenous polynucleotide may encode two or more suchmolecules, or a plurality of such molecules as may be desired. Whencombinational gene therapies are desired, two or more differentmolecules may be produced from a single rAAV expression system, oralternatively, a selected host cell may be transfected with two or moreunique rAAV expression systems, each of which may comprise one or moredistinct polynucleotides that encode a therapeutic agent.

In other embodiments, the invention also provides genetically-modifiedrAAV vectors that are comprised within an infectious adeno-associatedviral particle or a virion, or pluralities of such particles, whichthemselves may also be comprised within one or more diluents, buffers,physiological solutions or pharmaceutical vehicles, formulated foradministration to a mammal such as a human for therapeutic, and/orprophylactic gene therapy regimens. Such vectors, virus particles,virions, and pluralities thereof may also be provided in excipientformulations that are acceptable for veterinary administration toselected livestock, exotic or domesticated animals, companion animals(including pets and such like), as well as non-human primates,zoological or otherwise captive specimens, and such like, wherein theuse of such vectors and related gene therapy is indicated to produce abeneficial effect upon administration to such an animal.

The invention also concerns host cells that comprise at least one of thedisclosed rAAV vectors, virus particles, or virions. Such host cells areparticularly mammalian host cells, with human host cells beingparticularly highly preferred, and may be either isolated, in cell ortissue culture. In the case of genetically modified animal models, thetransformed host cells may even be comprised within the body of anon-human animal itself.

In certain embodiments, the creation of recombinant non-human hostcells, and/or isolated recombinant human host cells that comprise one ormore of the disclosed rAAV vectors is also contemplated to be useful fora variety of diagnostic, and laboratory protocols, including, forexample, means for the production of large-scale quantities of the rAAVvectors described herein. Such virus production methods are particularlycontemplated to be an improvement over existing methodologies includingin particular, those that require very high titers of the viral stocksin order to be useful as a gene therapy tool. The inventors contemplatethat one very significant advantage of the present methods will be theability to utilize lower titers of viral particles in mammaliantransduction protocols, yet still retain transfection rates at asuitable level.

Compositions comprising one or more of the disclosed rAAV vectors,expression systems, infectious AAV particles, or host cells also formpart of the present invention, and particularly those compositions thatfurther comprise at least a first pharmaceutically-acceptable excipientfor use in therapy, and for use in the manufacture of medicaments forthe treatment of one or more mammalian diseases, disorders,dysfunctions, or trauma. Such pharmaceutical compositions may optionallyfurther comprise one or more diluents, buffers, liposomes, a lipid, alipid complex; or the tyrosine-modified rAAV vectors may be comprisedwithin a microsphere or a nanoparticle. Pharmaceutical formulationssuitable for intramuscular, intravenous, or direct injection into anorgan or tissue or a plurality of cells or tissues of a human or othermammal are particularly preferred, however, the compositions disclosedherein may also find utility in administration to discreet areas of themammalian body, including for example, formulations that are suitablefor direct injection into one or more organs, tissues, or cell types inthe body. Such injection sites include, but are not limited to, thebrain, a joint or joint capsule, a synovium or subsynovium tissue,tendons, ligaments, cartilages, bone, peri-articular muscle or anarticular space of a mammalian joint, as well as direct administrationto an organ such as the heart, liver, lung, pancreas, intestine, brain,bladder, kidney, or other site within the patient's body, including, forexample, introduction of the viral vectors via intraabdominal,intrathorascic, intravascular, or intracerebroventricular delivery.

Other aspects of the invention concern recombinant adeno-associatedvirus virion particles, compositions, and host cells that comprise,consist essentially of, or consist of, one or more of the rAAV vectorsdisclosed herein, such as for example pharmaceutical formulations of thevectors intended for administration to a mammal through suitable means,such as, by intramuscular, intravenous, intra-articular, or directinjection to one or more cells, tissues, or organs of a selected mammal.Typically, such compositions may be formulated withpharmaceutically-acceptable excipients as described hereinbelow, and maycomprise one or more liposomes, lipids, lipid complexes, microspheres ornanoparticle formulations to facilitate administration to the selectedorgans, tissues, and cells for which therapy is desired.

Kits comprising one or more of the disclosed rAAV vectors, virions,viral particles, transformed host cells or pharmaceutical compositionscomprising such; and instructions for using the kit in a therapeutic,diagnostic, or clinical embodiment also represent preferred aspects ofthe present disclosure. Such kits may further comprise one or morereagents, restriction enzymes, peptides, therapeutics, pharmaceuticalcompounds, or means for delivery of the composition(s) to host cells, orto an animal (e.g., syringes, injectables, and the like). Such kits maybe therapeutic kits for treating, preventing, or ameliorating thesymptoms of a disease, deficiency, dysfunction, and/or injury, and maycomprise one or more of the modified rAAV vector constructs, expressionsystems, virion particles, or a plurality of such particles, andinstructions for using the kit in a therapeutic and/or diagnosticmedical regimen. Such kits may also be used in large-scale productionmethodologies to produce large quantities of the viral vectorsthemselves (with or without a therapeutic agent encoded therein) forcommercial sale, or for use by others, including e.g., virologists,medical professionals, and the like.

Another important aspect of the present invention concerns methods ofuse of the disclosed rAAV vectors, virions, expression systems,compositions, and host cells described herein in the preparation ofmedicaments for preventing, treating or ameliorating the symptoms ofvarious diseases, dysfunctions, or deficiencies in an animal, such as avertebrate mammal. Such methods generally involve administration to amammal, or human in need thereof, one or more of the disclosed vectors,virions, viral particles, host cells, compositions, or pluralitiesthereof, in an amount and for a time sufficient to prevent, treat, orlessen the symptoms of such a disease, dysfunction, or deficiency in theaffected animal. The methods may also encompass prophylactic treatmentof animals suspected of having such conditions, or administration ofsuch compositions to those animals at risk for developing suchconditions either following diagnosis, or prior to the onset ofsymptoms.

As described above, the exogenous polynucleotide will preferably encodeone or more proteins, polypeptides, peptides, ribozymes, or antisenseoligonucleotides, or a combination of these. In fact, the exogenouspolynucleotide may encode two or more such molecules, or a plurality ofsuch molecules as may be desired. When combinational gene therapies aredesired, two or more different molecules may be produced from a singlerAAV expression system, or alternatively, a selected host cell may betransfected with two or more unique rAAV expression systems, each ofwhich will provide unique heterologous polynucleotides encoding at leasttwo different such molecules.

In other embodiment, the invention also concerns the disclosed rAAVvectors comprised within an infectious adeno-associated viral particle,comprised within one or more pharmaceutical vehicles, and may beformulated for administration to a mammal such as a human fortherapeutic, and/or prophylactic gene therapy regimens. Such vectors mayalso be provided in pharmaceutical formulations that are acceptable forveterinary administration to selected livestock, domesticated animals,pets, and the like.

The invention also concerns host cells that comprise the disclosed rAAVvectors and expression systems, particularly mammalian host cells, withhuman host cells being particularly preferred.

Compositions comprising one or more of the disclosed rAAV vectors,expression systems, infectious AAV particles, host cells also form partof the present invention, and particularly those compositions thatfurther comprise at least a first pharmaceutically-acceptable excipientfor use in the manufacture of medicaments and methods involvingtherapeutic administration of such rAAV vectors. Such pharmaceuticalcompositions may optionally further comprise liposomes, a lipid, a lipidcomplex; or the rAAV vectors may be comprised within a microsphere or ananoparticle. Pharmaceutical formulations suitable for intramuscular,intravenous, or direct injection into an organ or tissue of a human areparticularly preferred.

Other aspects of the invention concern recombinant adeno-associatedvirus virion particles, compositions, and host cells that comprise oneor more of the AAV vectors disclosed herein, such as for examplepharmaceutical formulations of the vectors intended for administrationto a mammal through suitable means, such as, by intramuscular,intravenous, or direct injection to cells, tissues, or organs of aselected mammal. Typically, such compositions may be formulated withpharmaceutically-acceptable excipients as described hereinbelow, and maycomprise one or more liposomes, lipids, lipid complexes, microspheres ornanoparticle formulations to facilitate administration to the selectedorgans, tissues, and cells for which therapy is desired.

Kits comprising one or more of the disclosed vectors, virions, hostcells, viral particles or compositions; and (ii) instructions for usingthe kit in therapeutic, diagnostic, or clinical embodiments alsorepresent preferred aspects of the present disclosure. Such kits mayfurther comprise one or more reagents, restriction enzymes, peptides,therapeutics, pharmaceutical compounds, or means for delivery of thecompositions to host cells, or to an animal, such as syringes,injectables, and the like. Such kits may be therapeutic kits fortreating or ameliorating the symptoms of particular diseases, and willtypically comprise one or more of the modified AAV vector constructs,expression systems, virion particles, or therapeutic compositionsdescribed herein, and instructions for using the kit.

Another important aspect of the present invention concerns methods ofuse of the disclosed vectors, virions, expression systems, compositions,and host cells described herein in the preparation of medicaments fortreating or ameliorating the symptoms of various polypeptidedeficiencies in a mammal. Such methods generally involve administrationto a mammal, or human in need thereof, one or more of the disclosedvectors, virions, host cells, or compositions, in an amount and for atime sufficient to treat or ameliorate the symptoms of such a deficiencyin the affected mammal. The methods may also encompass prophylactictreatment of animals suspected of having such conditions, oradministration of such compositions to those animals at risk fordeveloping such conditions either following diagnosis, or prior to theonset of symptoms.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is an amino acid sequence of the capsid protein of thewild-type adeno-associated virus serotype 1 (AAV1);

SEQ ID NO:2 is an amino acid sequence of the capsid protein of thewild-type adeno-associated virus serotype 2 (AAV2);

SEQ ID NO:3 is an amino acid sequence of the capsid protein of thewild-type adeno-associated virus serotype 3 (AAV3);

SEQ ID NO:4 is an amino acid sequence of the capsid protein of thewild-type adeno-associated virus serotype 4 (AAV4);

SEQ ID NO:5 is an amino acid sequence of the capsid protein of thewild-type adeno-associated virus serotype 5 (AAV5);

SEQ ID NO:6 is an amino acid sequence of the capsid protein of thewild-type adeno-associated virus serotype 6 (AAV6);

SEQ ID NO:7 is an amino acid sequence of the capsid protein of thewild-type adeno-associated virus serotype 7 (AAV7);

SEQ ID NO:8 is an amino acid sequence of the capsid protein of thewild-type adeno-associated virus serotype 8 (AAV8);

SEQ ID NO:9 is an amino acid sequence of the capsid protein of thewild-type adeno-associated virus serotype 9 (AAV9);

SEQ ID NO:10 is an amino acid sequence of the capsid protein of thewild-type adeno-associated virus serotype 10 (AAV10);

SEQ ID NO:11 is an oligonucleotide primer sequence useful according tothe present invention; and

SEQ ID NO:12 is an oligonucleotide primer sequence useful according tothe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an exemplary schematic model for AAV2 trafficking inside amammalian host cell;

FIG. 2A-1, FIG. 2A-2, FIG. 2A-3, FIG. 2B-1, FIG. 2B-2, FIG. 2B-3, FIG.2C-1, FIG. 2C-2, FIG. 23-3, FIG. 2D-1 FIG. 2D-2, and FIG. 2D-3 showamino acid alignment of the wild-type AAV1-10 capsids. FIG. 2A-1 andFIG. 2A-2 show amino acid alignment of the wild-type AAV1-10 serotypecapsids (SEQ ID NO:1 through SEQ ID NO:10). FIG. 2B-I, FIG. 2B-2, andFIG. 2B-3 show conserved, surface-exposed lysine residues in thewild-type AAV1-12 capsids, as well as embodiments of amino acidmodifications. The lysine residues conserved among AAV1-12 are shown inbold. FIG. 2C-1 to FIG. 2C-3 show amino acid alignment of the wild-typeAAV1-10 serotype capsids, as well as surface-exposed tyrosine residuesthat are conserved among AAV1-10 capsids (conserved, surface-exposedresidues are shown in bold). FIG. 2D-1 to FIG. 2D-3 show amino acidalignment of the wild-type AAV1-10 serotype capsids, as well assurface-exposed serine and threonine residues that are conserved inamong AAV1-10 capsids (conserved, surface-exposed residues are shown inbold);

FIG. 3A and FIG. 3R show the effect of various kinase inhibitors onssAAV and scAAV mediated EGFP expression in HEK293 cells. Cells werepretreated with inhibitors for 1 hr before infection and then transducedwith 1×10³ vgs/cell. FIG. 3A: Transgene expression was detected byfluorescence microscopy 48 h post infection. FIG. 3B: Images from threevisual fields were analyzed as described herein. *P<0.005, **P<0.001 vs.WT AAV2;

FIG. 4A and FIG. 4B show an analysis of EGFP expression aftertransduction of 293 cells with individual site-directed AAV2 capsidmutants. Each of the 15 surface-exposed serines (S) in AAV2 capsid wassubstituted with valine (V) and evaluated for its efficiency to mediatetransgene expression. FIG. 4A: EGFP expression analysis at 48 hpost-infection at an MOI of 1×10³ vgs/cell. FIG. 4B: Quantitation oftransduction efficiency of each of the serine-mutant AAV2 vectors.*P<0.005, **P<0.001 vs. WT AAV2;

FIG. 5 shows packaging and transduction efficiencies of variousserine-valine mutant AAV2 vectors relative to wild-type (WT) AAV2vectors. Briefly, vector packaging and infectivity assays were performedat least twice for each of the mutant-AAV vectors. The packagingefficiency was determined by quantitative PCR analyses. The transductionefficiency was estimated by fluorescence intensity. *No fluorescence wasdetected at the MOI tested;

FIG. 6A and FIG. 6B show an evaluation of the effect of serinesubstitution at position 662 in the AAV2 capsid with different aminoacids in mediating transgene expression. The following 8 serine mutantswere generated with different amino acids: S662→Valine (V), S662→Alanine(A), S662→Asparagine (N), S662→Aspartic acid (D), S662→Histidine (H),S662→Isoleucine (I), S662→Leucine (L), and S662→Phenylalanine (F), andtheir transduction efficiency in 293 cells was analyzed. FIG. 6A: EGFPexpression analysis at 48-hr after infection of 293 cells at an MOT of1×10³ vgs/cell. FIG. 6B: Quantitation of the transduction efficiency ofeach of the serine-mutant AAV2 vectors. *P<0.005, **P<0.001 vs. WT AAV2;

FIG. 7 illustrates packaging and transduction efficiencies of exemplaryserine-mutant vectors in accordance with one aspect of the presentinvention which have been replaced with various amino acids relative towild-type (WT) AAV2 vector. The packaging and infectivity assays wereperformed as described herein. V=Valine; Λ=Alanine; D=Aspartic acid;F=Phenylalanine H=Histidine; N=Asparagine; L=Leucine; and I=Isoleucine;

FIG. 8A and FIG. 8B show an analysis of correlation of transductionefficiency of AAV2-S662V vectors with p38 MAPK activity in various celltypes. FIG. 8A: Quantitation of the transduction efficiency of WT- andS662V-AAV2 vectors in 293, HeLa, NIH3T3, H2.35 and moDCs. FIG. 8B:Western blot analysis of lysates from different cell lines for p-p38MAPK expression levels. Total p38 MAPK and GAPDH levels were measuredand used as loading controls. *P<0.005, **P<0.001 vs. WT AAV2;

FIG. 9A, FIG. 9B, and FIG. 9C demonstrate AAV vector-mediated transgeneexpression in monocytes-derived dendritic cells (moDCs) n accordancewith one aspect of the present invention. FIG. 9A shows the effect ofJNK and p38 MAPK inhibitors, and site-directed substitution of theserine residue at position 662 on EGFP expression. FIG. 9B providesquantitation of the data in FIG. 9A at 48-hr after infection andinitiation of maturation. FIG. 9C shows a representative analysis ofexpression of co-stimulatory markers such as CD80, CD83, CD86 in moDCsinfected with one particular vector in accordance with the presentinvention—AAV2-S662V—at an MOI 5×10⁴ vgs/cell. iDCs (immature dendriticcells), and mDCs (mature dendritic cells) stimulated with cytokines andgenerated as described herein were used as negative and positivecontrols, respectively. A representative example is shown. *P<0.005,**P<0.001 vs. WT AAV2.

FIG. 10 illustrates an analysis of hTERT-specific cytotoxicT-lymphocytes (CTLs) killing activity on K562 cells. CTLs were generatedafter transduction of moDCs by AAV2-S662V vectors encoding the truncatedhuman telomerase (hTERT). AAV2-S662V-EGFP vector-transduced moDCs wereused to generate non-specific CTLs. Pre-stained with3,3-diocladecyloxacarbocyanine (DiOC18(3)), a green fluorescent membranestain, 1×10⁵ target K562 cells were co-cultured overnight with differentratios of CTLs (in this illustrative example 80:1, 50:1, 20:1, 10:1, and5:1). Membrane-permeable nucleic acid counter-stain, propidium iodide,was added to label the cells with compromised plasma membranes.Percentages of killed, double stain-positive cells were analyzed by flowcytometry;

FIG. 11A and FIG. 11B demonstrate that site-directed mutagenesis ofsurface-exposed serine residues increased transduction efficiency of 293cells in exemplary scAAV vectors prepared in accordance with one aspectof the present invention;

FIG. 12A and FIG. 12B reveal that site-directed mutagenesis ofsurface-exposed threonine residues increased transduction efficiency of293 cells in exemplary scAAV vectors prepared in accordance with oneaspect of the present invention;

FIG. 13A and FIG. 13B illustrate that site-directed mutagenesis ofexemplary combinations of surface-exposed serine, threonine and/ortyrosine residues also significantly increased the transductionefficiency of H2.35 cells when particularly exemplary scAAV vectors wereprepared and used in accordance with the methods described herein;

FIG. 14A, FIG. 14B, FIG. 14C, FIG. 14D, and FIG. 14E demonstrate AAVvector-induced innate immune response in mice in vivo. FIG. 14A showsgene expression profiling of innate immune mediators was performed, anddata for fold changes in gene expression at the 2-hr time pointcomparing AAV vectors with Bay11 (hatched or open bars) with AAV vectorswithout Bay11 (black or grey bars) are shown. The minimal thresholdfold-increase (horizontal black line) was 2.5. FIG. 14B shows a westernblot analysis of liver homogenates from mice at 9-hrs followingmock-injections, or injections with scAAV vectors, with and withoutprior administration of Bay11. Samples were analyzed by using anti-pS2antibody for detection of NF-KB signaling in response to AAV exposure.Anti-β-actin antibody was used as a loading control. FIG. 14C shows thehumoral response to exemplary AAV vectors in the absence or presence ofNF-KB inhibitor. Anti-AAV2 IgG2a levels were determined in peripheralblood from mice at Day 10 following injections with scAAV vectors, withand without prior administration of Bay11 (n=4 each). FIG. 14D shows thetransgene expression in murine hepatocytes 10 days' post-injection of1×10¹¹ vgs each of WT-scAAV-EGFP or TM-scAAV-EFGP vectors/animal via thetail-vein. FIG. 14E illustrates the quantitative analyses ofrepresentative data from the study depicted in FIG. 14D;

FIG. 15A, FIG. 15B, FIG. 15C, and FIG. 15D illustrate AAV3-mediatedtransgene expression in T47D and T47D+hHGFR cells. FIG. 15A: Equivalentnumbers of T47D and T47D+hHGFR cells were infected with variousindicated multiplicity-of-infection of scAAV3-CBΛp-EGFP vectors underidentical conditions. Transgene expression was determined byfluorescence microscopy 72 hrs post-infection. FIG. 15B: T47D+hHGFRcells were transduced with 2,000 vgs/cell of scAAV3 vectors—either inthe absence or presence of 5 μg/mL of hHGF. Transgene expression wasdetermined as above. FIG. 15C: The effect of HGFR kinase-specificinhibitor, BMS-777607 (BMS), on AAV3-mediated transgene expression isshown. T47D and T47D+hHGFR cells were mock-treated or pretreated withBMS for 2 hs. Whole-cell lysates were prepared and analyzed on Westernblots using various indicated primary antibodies. β-actin was used as aloading control. FIG. 15D: Transduction efficiency of WT and single,double, and triple tyrosine-mutant AAV3 vectors is shown. Huh7 cellswere transduced with WT or various indicated Y-F mutant scAAV3-CBAp-EGFPvectors under identical conditions;

FIG. 16 shows the quantification of transgene expression by AAV2-K544F,AAV2-K556E, AAV2-K544/566E, and AAV2-Y444/500/730F;

FIG. 17A, FIG. 17B, FIG. 17C, FIG. 17D and FIG. 17E illustrate thetransduction efficiency of WT- and lysine-mutant scAAV8 vectors inprimary hepatocytes in vivo (C57BL/6 mice; 1×10¹⁰ scAAV-2-CBAp-Flucvectors; tail-vein injections; 2-weeks) (experiment 2);

FIG. 18 demonstrates the quantification of transgene expression byAAV8-K530E, AAV8-K547E, and AAV8-K569E in accordance with one aspect ofthe present invention.

FIG. 19A and FIG. 19B shows the transduction efficiency of WT- andlysine-mutant scAAV2 vectors in primary hepatocytes in vive (C57BL/6mice; 1×10¹⁰ scAAV-2-CBAp-EGFP vectors; tail-vein injections; 2-weeks);

FIG. 20A and FIG. 20B show the transduction efficiency of WT- andexemplary lysine- or tyrosine-substituted mutant scAAV2 vectors inaccordance with one aspect of the present invention in a study involvingprimary hepatocytes in vivo (C57BL/6 mice; 1×10¹⁰ scAAV-2-CBAp-Flucvectors; tail-vein injections; 2-weeks);

FIG. 21A, FIG. 21B, FIG. 21C, FIG. 21D, FIG. 21E, FIG. 21F, FIG. 21G,FIG. 21H, and FIG. 21I show the transduction efficiency of WT- andexemplary lysine-mutated capsid-containing scAAV2 vectors in HeLa cellsin vitro (2,000 vgs/cell; 48 hrs) in accordance with one aspect of thepresent invention;

FIG. 22A, FIG. 22B, FIG. 22C, FIG. 22D, FIG. 22E, FIG. 22F, FIG. 22G,FIG. 22H, and FIG. 22I show that site-directed mutagenesis of acombination of surface-exposed serine, threonine and/or tyrosineresidues increase transduction efficiency of monocyte-derived dendriticcells by scAAV vectors in accordance with one aspect of the presentinvention;

FIG. 23A, FIG. 23B, FIG. 23C, FIG. 23D, FIG. 23E, and FIG. 23F showtransduction efficiency of exemplary AAV2 lysine mutants prepared inaccordance with the methods disclosed herein in HeLa and HEK293 cells invitro (MOI 2000). The relative fold-increase in gene expression is shownas inserts;

FIG. 24 shows the transduction efficiency of WT- and exemplarylysine-mutated capsid-expressing scAAV8 vectors in murine hepatocytes invivo (C57BL/6 mice; 1×10¹⁰ scAAV-2-CBAp-Fluc vectors; tail-veininjections; 2-weeks) (experiment 1); and

FIG. 25 shows the quantification of transgene expression by exemplaryAAV8 vectors expressing particular lysine-substituted capsid proteinmutants. Shown are the representative constructs AAV8-K530E, AAV8-K547E,and AAV8-K569E.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention provides AAV capsid proteins comprisingmodification of one or a combination of the surface-exposed lysine,serine, threonine and/or tyrosine residues in the VP3 region. Alsoprovided are rAAV virions that include one or more of the AAV capsidprotein mutations disclosed herein, as well as nucleic acid moleculesand rAAV vectors encoding the AAV capsid proteins of the presentinvention. Advantageously, the rAAV vectors and virions of the presentinvention have improved efficiency in transduction of a variety ofcells, tissues and organs of interest and/or reduces host immuneresponses to the vectors, when compared to wild-type rAAV vectors andvirions.

In one embodiment, the present invention provides a nucleic acidmolecule comprising a nucleotide sequence encoding an AAV capsidprotein, wherein the VP3 region of the AAV capsid protein comprises anon-lysine residue at one or more positions that correspond to one ormore lysine residues in the VP3 region of the capsid protein of thewild-type AAV [e.g., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:5, SEQ ID NO:6, SEQ TD NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQID NO:10; and in one particularly exemplary embodiment, the capsidprotein of wild-type AAV2 (SEQ ID NO:2)], wherein one or more of thelysine residue(s) in the VP3 region of the wild-type AAV is selectedfrom the one or more lysine residues as illustrated in FIG. 2A and FIG.2B.

In a specific embodiment, one or more surface-exposed lysine residuecorresponding to one or more lysine residues selected from the groupconsisting of K258, K321, K459, K490, K507, K527, K572, K532, K544,K549, K556, K649, K655, K665, and K706 of the wild-type AAV capsidprotein [e.g., SEQ ID NOs:1-10; in one embodiment, the capsid protein ofwild-type AAV2 (SEQ ID NO:2)] are modified.

In one embodiment, the surface-exposed lysine residue corresponding toK532 of the wild-type AAV2 capsid sequence is modified. In oneembodiment, the surface-exposed lysine residue of the AAV capsid ismodified into glutamic acid (E) or arginine (R). In one specificembodiment, the surface-exposed lysine residue corresponding to K532 ofthe wild-type AAV2 capsid sequence is modified into arginine (K532R).

In certain embodiments, one or more surface-exposed lysine residuescorresponding to K490, K544, K549, and K556 of the wild-type AAV2 capsidsequence are modified. In certain specific embodiments, one or moresurface-exposed lysine residue corresponding K490, K544, K549, and K556of the wild-type AAV2 capsid sequence are modified into glutamic acid(E).

In one embodiment, the present invention provides AAV2 vectors whereinsurface-exposed lysine residues corresponding to K544 and K556 residuesof the wild-type AAV2 capsid are modified into glutamic acid (E).

In certain embodiments, one or more surface-exposed lysine residuescorresponding to K530, K547, and K569 of the wild-type AAV8 capsidsequence are modified. In certain specific embodiments, one or moresurface-exposed lysine residue corresponding K530, K547, and K569 of thewild-type AAV2 capsid sequence are modified into glutamic acid (E) orarginine (R).

In addition, the present invention provides a method for transduction ofcells, tissues, and/or organs of interest, comprising introducing into acell, a composition comprising an effective amount of a rAAV vectorand/or virion of the present invention.

Phosphorylation of surface-exposed lysine, serine, threonine and/ortyrosine residues on the AAV capsids can result in theubiquitination/proteasomal degradation of the vectors. Serine/threonineprotein kinases are involved in a wide variety of cellular processesincluding cell differentiation, transcription regulation, anddevelopment. Phosphorylation of the surface-exposed serine and/orthreonine residues on the viral capsid induces proteasome-mediateddegradation of the vectors and reduces vector transduction efficiency.Cellular epidermal growth factor receptor protein tyrosine kinase(EGFR-PTK) also phosphorylates capsids at surface tyrosine residues,and, thus negatively impacts nuclear transport and subsequent transgeneexpression by recombinant AAV2 vectors.

Surface-exposed lysine, serine, threonine and/or tyrosine residues onthe AAV capsids are identified (FIG. 2A, FIG. 2B, FIG. 2C, and FIG. 21).For instance, the VP3 region of the capsid protein of the wild-type AAV2contains various surface-exposed lysine (K) residues (K258, K321, K459,K490, K507, K527, K572, K532, K544, K549, K556, K649, K655, K665, K706),surface-exposed serine (S) residues (S261, S264, S267, S276, S384, S458,S468, S498, S578, S658, S662, S668, S707, 5721), surface-exposedthreonine (T) residues (T251, T329, T330, T454, T455, T503, T550, T592,T581, T597, T491, T671, T659, T660, T701, T713, T716), andsurface-exposed tyrosine residues (Y252, Y272, Y444, Y500, Y700, Y704,Y730). As shown in FIG. 2A, FIG. 2B, FIG. 2C, and FIG. 2D, thesesurface-exposed lysine, serine, threonine and/or tyrosine residues ofthe capsids are highly conserved among AAV1-12.

Site-directed mutagenesis of the surface-exposed lysine, serine,threonine, and/or tyrosine residues was performed and the results showthat modification or substitution of one or a combination of thesurface-exposed residues can enable the AAV vector to bypass theubiquitination and proteasome-mediated degradation steps, therebyyielding novel AAV vectors with high-efficiency transduction.Substitution of surface exposed tyrosine residues on AAV capsids permitsthe vectors to escape ubiquitination, and thus, inhibitsproteasome-mediated degradation. Although phosphorylated AAV vectorscould enter cells as efficiently as their unphosphorylated counterparts,their transduction efficiency was significantly reduced. This reductionwas not due to impaired viral second-strand DNA synthesis sincetransduction efficiency of both single-stranded AAV (ssAAV) andself-complementary AAV (rAAV) vectors was decreased.

Recombinant AAV vectors containing point mutations in surface exposedtyrosine residues confer higher transduction efficiency at lower doses,when compared to the wild-type (WT) AAV vectors.

In addition, in accordance of the present invention, (i) site-directedmutagenesis of the 15 surface-exposed serine (S) residues on the AAV2capsid with valine (V) residues leads to improved transductionefficiency of S458V, S492V, and S662V mutant vectors compared with theWT AAV2 vector; (ii) the S662V mutant vector efficiently transducesprimary human monocyte-derived dendritic cells (moDCs), a cell type notreadily amenable to transduction by the conventional AAV vectors; (iii)high-efficiency transduction of moDCs by S662V mutant does not induceany phenotypic changes in these cells; and (iv) recombinant S662V-rAAVvectors carrying a truncated human telomerase (hTERT) gene transducedDCs result in rapid, specific T-cell clone proliferation and generationof robust CTLs, which lead to specific cell lysis of K562 cells. Theresults demonstrate that the serine-modified rAAV2 vectors of thepresent invention result in high-efficiency transduction of moDCs.

In the setting of tumor immunotherapy, the time of T-cell activation andthe potency and longevity of CD8 T cell responses are crucial factors indetermining the therapeutic outcome. In accordance with the presentinvention, increased transduction efficiency of moDC by theserine-mutant ΛΛV2 vectors resulted in superior priming of T-cells.Human telomerase was used as a specific target since clinical studieshave shown that human telomerase is an attractive candidate for abroadly expressed rejection antigen for many cancer patients. Inaddition, transduction efficiency of the S662V mutant vector was furtheraugmented by pre-treatment of cells with specific inhibitors of JNK andp38 MAPK, indicating that one or more surface-exposed threonine (T)residues on AAV2 capsids are most likely phosphorylated by thesekinases.

Recombinant AAV Vectors and Virions

One aspect of the invention provides AAV capsid proteins comprisingmodification of one or a combination of the surface-exposed lysine,serine, threonine and/or tyrosine residues in the VP3 region. Alsoprovided are rAAV virions comprising the AAV capsid proteins of thepresent invention, as well as nucleic acid molecules and rAAV vectorsencoding the AAV capsid proteins of the present invention.Advantageously, the rAAV vectors and virions of the present inventionhave improved efficiency in transduction of a variety of cells, tissuesand organs of interest, when compared to wild-type rAAV vectors andvirions.

In one embodiment, the present invention provides a nucleic acidmolecule comprising a nucleotide sequence encoding an AAV capsidprotein, wherein the AAV capsid protein comprises modification of one ora combination of the surface-exposed lysine, serine, threonine and/ortyrosine residues in the VP3 region. Advantageously, modification of thesurface-exposed lysine, serine, threonine and/or tyrosine residuesprevents or reduces the level of ubiquitination of the AAV vectors, and,thereby, prevents or reduces the level of proteasome-mediateddegradation. In addition, modification of the surface-exposed lysine,serine, threonine and/or tyrosine residues in accordance with thepresent invention improves transduction efficiency.

In one embodiment, the nucleic acid molecule comprising a nucleotidesequence encoding an AAV capsid protein, wherein the VP3 region of theAAV capsid protein comprises one or a combination of the followingcharacteristics:

(a)

(i) a non-lysine residue at one or more positions that correspond to alysine residue in the VP3 region of the capsid protein of the wild-typeAAV (e.g., SEQ ID NOs:1-10; in one embodiment, the capsid protein ofwild-type AAV2 (SEQ ID NO:2)), wherein said lysine residue in the VP3region of the wild-type AAV is selected from the group consisting ofK258, K321, K459, K490, K507, K527, K572, K532, K544, K549, K556, K649,K655, K665, and K706, wherein said non-lysine residue does not result inphosphorylation and/or ubiquitination of an AAV vector; and/or

(ii) a chemically-modified lysine residue at one or more positions thatcorrespond to a lysine residue in the VP3 region of the capsid proteinof the wild-type AAV (e.g., SEQ ID NOs:1-10; in one embodiment, thecapsid protein of wild-type AAV2 (SEQ ID NO:2)), wherein said lysineresidue in the VP3 region of the wild-type AAV is selected from thegroup consisting of K258, K321, K459, K490, K507, K527, K572, K532,K544, K549, K556, K665, K649, K655, and K706, wherein saidchemically-modified lysine residue does not result in phosphorylationand/or ubiquitination of an AAV vector,

(iii) a non-lysine residue at one or more positions that correspond to alysine residue in the VP3 region of the capsid protein of the wild-typeAAV (e.g., SEQ ID NOs:1-10; in one embodiment, the capsid protein ofwild-type AAV8 (SEQ ID NO:8)), wherein said lysine residue in the VP3region of the wild-type AAV is selected from the group consisting ofK530, K547, and K569, wherein said non-lysine residue does not result inphosphorylation and/or ubiquitination of an AAV vector; and/or

(iv) a chemically-modified lysine residue at one or more positions thatcorrespond to a lysine residue in the VP3 region of the capsid proteinof the wild-type AAV (e.g., SEQ ID NOs:1-10; in one embodiment, thecapsid protein of wild-type AAV8 (SEQ ID NO:8)), wherein said lysineresidue in the VP3 region of the wild-type AAV is selected from thegroup consisting of K530, K547, and K569, wherein saidchemically-modified lysine residue does not result in phosphorylationand/or ubiquitination of an AAV vector;

(b)

(i) a non-serine residue at one or more positions that correspond to aserine residue in the VP3 region of the capsid protein of the wild-typeAAV (e.g., SEQ ID NOs:1-10; in one embodiment, the capsid protein ofwild-type AAV2 (SEQ ID NO:2)), wherein said serine residue in the VP3region of the wild-type AAV is selected from the group consisting ofS261, S264, S267, S276, S384, S458, S468, S498, S658, S662, S668, S707,and S721, wherein said non-serine residue does not result inphosphorylation and/or ubiquitination of an AAV vector; and/or

(ii) a chemically-modified serine residue at one or more positions thatcorrespond to a serine residue in the VP3 region of the capsid proteinof the wild-type AAV (e.g., SEQ ID NOs:1-10; in one embodiment, thecapsid protein of wild-type AAV2 (SEQ ID NO:2)), wherein said serineresidue in the VP3 region of the wild-type AAV is selected from thegroup consisting of S261, S264, S267, S276, S384, S458, S492, S498,S578, S658, S662, S668, S707, and 5721, wherein said chemically-modifiedserine residue does not result in phosphorylation and/or ubiquitinationof an AAV vector;

(c)

(i) a non-threonine residue at one or more positions that correspond toa threonine residue in the VP3 region of the capsid protein of thewild-type AAV (e.g., SEQ ID NOs:1-10; in one embodiment, the capsidprotein of wild-type AAV2 (SEQ ID NO:2)), wherein said threonine residuein the VP3 region of the wild-type AAV is selected from the groupconsisting of T251, T329, T330, T454, T455, T503, T550, T592, T581,T597, T491, T671, T659, T660, T701, T713, and T716, wherein saidnon-threonine residue does not result in phosphorylation and/orubiquitination of an AAV vector; and/or

(ii) a chemically-modified threonine residue at one or more positionsthat correspond to a threonine residue in the VP3 region of the capsidprotein of the wild-type AAV (e.g., SEQ ID NOs:1-10; in one embodiment,the capsid protein of wild-type AAV2 (SEQ ID NO:2)), wherein saidthreonine residue in the VP3 region of the wild-type AAV is selectedfrom the group consisting of T251, T329, T330, T454, T455, T503, T550,T592, T581, T597, T491, T671, T659, T660, T701, T713, and T716, whereinsaid chemically-modified threonine residue does not result inphosphorylation and/or ubiquitination of an AAV vector; and/or

(d)

(i) a non-tyrosine residue at one or more positions that correspond to atyrosine residue in the VP3 region of the capsid protein of thewild-type AAV (e.g., SEQ ID NOs:1-10; in one embodiment, the capsidprotein of wild-type AAV2 (SEQ ID NO:2)), wherein said tyrosine residuein the VP3 region of the wild-type AAV is selected from the groupconsisting of Y252, Y272, Y444, Y500, Y700, Y704, and Y730, wherein saidnon-tyrosine residue does not result in phosphorylation and/orubiquitination of an AAV vector, and/or

(ii) a chemically-modified tyrosine residue at one or more positionsthat correspond to a tyrosine residue in the VP3 region of the capsidprotein of the wild-type AAV (e.g., SEQ ID NOs:1-10; in one embodiment,the capsid protein of wild-type AAV2 (SEQ ID NO:2)), wherein saidtyrosine residue in the VP3 region of the wild-type AAV is selected fromthe group consisting of Y252, Y272, Y444, Y500, Y700, Y704, and Y730,wherein said chemically-modified tyrosine residue does not result inphosphorylation and/or ubiquitination of an AAV vector.

In another embodiment, the present invention provides a nucleic acidmolecule comprising a nucleotide sequence encoding an AAV capsidprotein, wherein one or more of surface-exposed lysine, serine,threonine and/or tyrosine residues in the VP3 region are modified asfollows:

(a)

(i) at least one lysine residue in the VP3 region is chemically modifiedor is modified into a non-lysine residue, wherein the modified residuecorresponds to K258, K321, K459, K490, K507, K527, K572, K532, K544,K549, K556, K649, K655, or K706 of the wild-type AAV capsid sequence(e.g., SEQ ID NOs:1-10; in one embodiment, the capsid protein ofwild-type AAV2 (SEQ ID NO:2)), wherein said non-lysine residue or saidchemically-modified lysine residue does not result in phosphorylationand/or ubiquitination of an AAV vector; and/or

(ii)

at least one lysine residue in the VP3 region is chemically modified oris modified into a non-lysine residue, wherein the modified residuecorresponds to K530, K547, or K569 of the wild-type AAV capsid sequence(e.g., SEQ ID NOs:1-10; in one embodiment, the capsid protein ofwild-type AAV8 (SEQ ID NO:8)), wherein said non-lysine residue or saidchemically-modified lysine residue does not result in phosphorylationand/or ubiquitination of an AAV vector;

(b) at least one serine residue in the VP3 region is chemically modifiedor is modified into a non-serine residue, wherein the modified residuecorresponds to S261, S264, S267, S276, S384, S458, S468, S492, S498,S578, S658, S662, S668, S707, or S721 of the wild-type AAV capsidsequence (e.g., SEQ ID NOs:1-10; in one embodiment, the capsid proteinof wild-type AAV2 (SEQ ID NO:2)), wherein said non-serine residue orsaid chemically-modified serine residue does not result inphosphorylation and/or ubiquitination of an AAV vector;

(c) at least one threonine residue in the VP3 region is chemicallymodified or is modified into a non-threonine residue, wherein themodified residue corresponds to T251, T329, T330, T454, T455, T503,T550, T592, T581, T597, T491, T671, T659, T660, T701, T713, or T716 ofthe wild-type AAV capsid sequence (e.g., SEQ ID NOs:1-10; in oneembodiment, the capsid protein of wild-type AAV2 (SEQ ID NO:2)), whereinsaid non-threonine residue or said chemically-modified threonine residuedoes not result in phosphorylation and/or ubiquitination of an AAVvector, and

(d) at least one tyrosine residue in the VP3 region is chemicallymodified or is modified into a non-tyrosine residue, wherein themodified residue corresponds to Y252, Y272, Y444, Y500, Y700, Y704, orY730 the wild-type AAV capsid sequence (e.g., SEQ ID NOs:1-10; in oneembodiment, the capsid protein of wild-type AAV2 (SEQ ID NO:2)), whereinsaid non-tyrosine residue or said chemically-modified tyrosine residuedoes not result in phosphorylation and/or ubiquitination of an AAVvector.

The present invention also provides AAV VP3 capsid proteins havingmodification of one or more surface-exposed lysine, serine, threonineand/or tyrosine residues. In one embodiment, the present inventionprovides an AAV VP3 capsid protein comprising one or a combination ofthe following characteristics:

(a)

(i) a non-lysine residue at one or more positions that correspond to alysine residue in the VP3 region of the capsid protein of the wild-typeAAV (e.g., SEQ ID NOs:1-10; in one embodiment, the capsid protein ofwild-type AAV2 (SEQ ID NO:2)), wherein said lysine residue in the VP3region of the wild-type AAV is selected from the group consisting ofK258, K321, K459, K490, K507, K527, K572, K532, K544, K549, K556, K649,K655, K665, and K706, wherein said non-lysine residue does not result inphosphorylation and/or ubiquitination of an AAV vector; and/or

(ii) a chemically-modified lysine residue at one or more positions thatcorrespond to a lysine residue in the VP3 region of the capsid proteinof the wild-type AAV (e.g., SEQ ID NOs:1-10; in one embodiment, thecapsid protein of wild-type AAV2 (SEQ ID NO:2)), wherein said lysineresidue in the VP3 region of the wild-type AAV is selected from thegroup consisting of K258, K321, K459, K490, K507, K527, K572, K532,K544, K549, K556, K649, K655, K665, and K706, wherein saidchemically-modified lysine residue does not result in phosphorylationand/or ubiquitination of an AAV vector;

(iii) a non-lysine residue at one or more positions that correspond to alysine residue in the VP3 region of the capsid protein of the wild-typeAAV (e.g., SEQ ID NOs:1-10; in one embodiment, the capsid protein ofwild-type AAV8 (SEQ ID NO:8)), wherein said lysine residue in the VP3region of the wild-type AAV is selected from the group consisting ofK530, K547, and K569, wherein said non-lysine residue does not result inphosphorylation and/or ubiquitination of an AAV vector; and/or

(iv) a chemically-modified lysine residue at one or more positions thatcorrespond to a lysine residue in the VP3 region of the capsid proteinof the wild-type AAV (e.g., SEQ ID NOs:1-10; in one embodiment, thecapsid protein of wild-type AAV8 (SEQ ID NO:8)), wherein said lysineresidue in the VP3 region of the wild-type AAV is selected from thegroup consisting of K530, K547, and K569, wherein saidchemically-modified lysine residue does not result in phosphorylationand/or ubiquitination of an AAV vector;

(b)

(i) a non-serine residue at one or more positions that correspond to aserine residue in the VP3 region of the capsid protein of the wild-typeAAV (e.g., SEQ ID NOs:1-10; in one embodiment, the capsid protein ofwild-type AAV2 (SEQ ID NO:2)), wherein said serine residue in the VP3region of the wild-type AAV is selected from the group consisting ofS261, S264, S276, S384, S458, S492, S578, S658, S662, S668, S707, and5721, wherein said non-serine residue does not result in phosphorylationand/or ubiquitination of an AAV vector; and/or

(ii) a chemically-modified serine residue at one or more positions thatcorrespond to a serine residue in the VP3 region of the capsid proteinof the wild-type AAV (e.g., SEQ ID NOs:1-10; in one embodiment, thecapsid protein of wild-type AAV2 (SEQ ID NO:2)), wherein said serineresidue in the VP3 region of the wild-type AAV is selected from thegroup consisting of S261, S264, S267, S276, S384, S468, 5492, 5498,S578, S658, S662, S707, and S721, wherein said chemically-modifiedserine residue does not result in phosphorylation and/or ubiquitinationof an AAV vector;

(c)

(i) a non-threonine residue at one or more positions that correspond toa threonine residue in the VP3 region of the capsid protein of thewild-type AAV (e.g., SEQ ID NOs:1-10; in one embodiment, the capsidprotein of wild-type AAV2 (SEQ ID NO:2)), wherein said threonine residuein the VP3 region of the wild-type AAV is selected from the groupconsisting of T251, T329, T330, T454, T455, T503, T550, T592, T581,T597, T491, T671, T659, T660, T701, T713, and T716, wherein saidnon-threonine residue does not result in phosphorylation and/orubiquitination of an AAV vector; and/or

(ii) a chemically-modified threonine residue at one or more positionsthat correspond to a threonine residue in the VP3 region of the capsidprotein of the wild-type AAV (e.g., SEQ ID NOs:1-10; in one embodiment,the capsid protein of wild-type AAV2 (SEQ ID NO:2)), wherein saidthreonine residue in the VP3 region of the wild-type AAV is selectedfrom the group consisting of T251, T329, T330, T454, T455, T503, T550,T592, T581, T597, T491, T671, T659, T660, T701, T713, and T716, whereinsaid chemically-modified threonine residue does not result inphosphorylation and/or ubiquitination of an AAV vector; and/or

(d)

(i) a non-tyrosine residue at one or more positions that correspond to atyrosine residue in the VP3 region of the capsid protein of thewild-type AAV (e.g., SEQ ID NOs:1-10; in one embodiment, the capsidprotein of wild-type AAV2 (SEQ ID NO:2)), wherein said tyrosine residuein the VP3 region of the wild-type AAV is selected from the groupconsisting of Y252, Y272, Y444, Y500, Y700, Y704, and Y730, wherein saidnon-tyrosine residue does not result in phosphorylation and/orubiquitination of an AAV vector; and/or

(ii) a chemically-modified tyrosine residue at one or more positionsthat correspond to a tyrosine residue in the VP3 region of the capsidprotein of the wild-type AAV (e.g., SEQ ID NOs:1-10; in one embodiment,the capsid protein of wild-type AAV2 (SEQ ID NO:2)), wherein saidtyrosine residue in the VP3 region of the wild-type AAV is selected fromthe group consisting of Y252, Y272, Y444, Y500, Y700, Y704, and Y730,wherein said chemically-modified tyrosine residue does not result inphosphorylation and/or ubiquitination of an AAV vector.

In another embodiment, the present invention provides an AAV capsidprotein, wherein one or more of surface-exposed lysine, serine,threonine and/or tyrosine residues in the VP3 region are modified asfollows:

(a)

(i) at least one lysine residue in the VP3 region is chemically modifiedor is modified into a non-lysine residue, wherein the modified residuecorresponds to K258, K321, K459, K490, K507, K527, K572, K532, K544,K549, K556, K649, K655, K665, or K706 of the wild-type AAV capsidsequence (e.g., SEQ ID NOs:1-10; in one embodiment, the capsid proteinof wild-type AAV2 (SEQ ID NO:2)), wherein said non-lysine residue orsaid chemically-modified lysine residue does not result inphosphorylation and/or ubiquitination of an AAV vector; and/or

(ii) at least one lysine residue in the VP3 region is chemicallymodified or is modified into a non-lysine residue, wherein the modifiedresidue correspond to K530, K547, or K569 of the wild-type AAV capsidsequence (e.g., SEQ ID NOs:1-10; in one embodiment, the capsid proteinof wild-type AAV8 (SEQ ID NO:8)), wherein said non-lysine residue orsaid chemically-modified lysine residue does not result inphosphorylation and/or ubiquitination of an AAV vector,

(b) at least one serine residue in the VP3 region is chemically modifiedor is modified into a non-serine residue, wherein the modified residuecorrespond to S261, S264, S267, S276, S384, S458, S468, S492, S498,S578, S658, S662, S668, S707, or S721 of the wild-type AAV capsidsequence (e.g., SEQ ID NOs:1-10; in one embodiment, the capsid proteinof wild-type AAV2 (SEQ ID NO:2)), wherein said non-serine residue orsaid chemically-modified serine residue does not result inphosphorylation and/or ubiquitination of an AAV vector;

(c) at least one threonine residue in the VP3 region is chemicallymodified or is modified into a non-threonine residue, wherein themodified residue correspond to T251, T329, T330, T454, T455, T503, T550,T592, T581, T597, T491, T671, T659, T660, T701, T713, or T716 of thewild-type AAV capsid sequence (e.g., SEQ ID NOs:1-10; in one embodiment,the capsid protein of wild-type AAV2 (SEQ ID NO:2)), wherein saidnon-threonine residue or said chemically-modified threonine residue doesnot result in phosphorylation and/or ubiquitination of an AAV vector;and

(d) at least one tyrosine residue in the VP3 region is chemicallymodified or is modified into a non-tyrosine residue, wherein themodified residue correspond to Y252, Y272, Y444, Y500, Y700, Y704, orY730 the wild-type AAV capsid sequence (e.g., SEQ ID NOs:1-10; in oneembodiment, the capsid protein of wild-type AAV2 (SEQ ID NO:2)), whereinsaid non-tyrosine residue or said chemically-modified tyrosine residuedoes not result in phosphorylation and/or ubiquitination of an AAVvector. As shown in FIG. 2A, FIG. 2B, FIG. 2C, and FIG. 2D,surface-exposed lysine, serine, threonine and/or tyrosine residueslocated in the VP3 region of the capsid protein are highly conservedamong various AAV serotypes (AAV1 to 12). In one embodiment, the nucleicacid molecule comprising a nucleotide sequence encoding an AAV capsidprotein, wherein the AAV serotype is selected from AAV1 to 12. Incertain embodiments, the wild-type AAV capsid protein has an amino acidsequence selected from SEQ ID NOs: 1-10.

In one specific embodiment, the nucleic acid molecule comprises anucleotide sequence encoding an AAV2 capsid protein. Theadeno-associated virus 2 (AAV2) is a non-pathogenic human parvovirus.Recombinant AAV2 vectors have been shown to transduce a wide variety ofcells and tissues in vitro and in vivo, and are currently in use inPhase I/II clinical trials for gene therapy of a number of diseases suchas cystic fibrosis, alpha-1 antitrypsin deficiency, Parkinson's disease,Batten's disease, and muscular dystrophy.

In one embodiment, the present invention provides an AAV capsid protein,wherein the AAV capsid protein comprises the amino acid sequence of thecapsid protein of the wild-type AAV2 (SEQ ID NO:2) except that one ormore of the amino acid residues of the wild-type AAV2 capsid aremodified as follows:

(a) at least one lysine residue of the wild-type AAV2 capsid sequenceselected from the group consisting of K258, K321, K459, K490, K507,K527, K572, K532, K544, K549, K556, K649, K655, K665, and K706 ismodified into a non-lysine residue, or said lysine residue is chemicallymodified so that said non-lysine residue or said chemically-modifiedlysine residue does not result in phosphorylation and/or ubiquitinationof an AAV vector;

(b) at least one serine residue of the wild-type AAV2 capsid sequenceselected from the group consisting of S261, S264, S267, S384, S458,S468, S492, S498, S578, S658, S662, S668, S707, and S721 is modifiedinto a non-serine residue, or said serine residue is chemically modifiedso that said non-serine residue or said chemically-modified serineresidue does not result in phosphorylation and/or ubiquitination of anAAV vector;

(c) at least one threonine residue of the wild-type AAV2 capsid sequenceselected from the group consisting of T251, T329, T330, T454, T455,T503, T550, T592, T581, T597, T491, T671, T659, T660, T701, T713, andT716 is modified into a non-threonine residue, or said threonine residueis chemically modified so that said non-threonine residue or saidchemically-modified threonine residue does not result in phosphorylationand/or ubiquitination of an AAV vector; and/or

(d) at least one tyrosine residue of the wild-type AAV2 capsid sequenceselected from the group consisting of Y252, Y272, Y444, Y500, Y700,Y704, and Y730 is modified into a non-threonine residue is modified intoa non-tyrosine residue, or said tyrosine residue is chemically modifiedso that said non-tyrosine residue or said chemically-modified tyrosineresidue does not result in phosphorylation and/or ubiquitination of anAAV vector. In one embodiment, a surface-exposed lysine residuecorresponding to a lysine residue selected from K532, K459, K490, K544,K549, K556, K527, K490, K143, or K137 of the wild-type AAV capsidsequence (e.g., SEQ ID NOs:1-10; in one embodiment, the capsid proteinof wild-type AAV2 (SEQ ID NO:2)) is modified into a non-lysine residueand/or is chemically modified so that said non-lysine residue or saidmodified lysine residue does not result in phosphorylation and/orubiquitination of an AAV vector.

In another embodiment, the present invention provides an AAV capsidprotein, wherein the AAV capsid protein comprises the amino acidsequence of the capsid protein of the wild-type AAV8 (SEQ ID NO:8)except that one or more surface-exposed lysine residues corresponding toK530, K547, and K569 of the wild-type AAV8 capsid are modified into anon-lysine residue (such as, glutamic acid (E), arginine (R)) and/or aremodified chemically modified, wherein said non-lysine residue or saidmodified lysine residue does not result in phosphorylation and/orubiquitination of an AAV vector. In certain embodiments, thesurface-exposed lysine residues of AAV sequence are modified intoglutamic acid (E), arginine (R), serine (S), or isoleucine (I) to avoidin phosphorylation and/or ubiquitination of the AAV vector.

The present invention also provides a nucleic acid molecule comprises anucleotide sequence encoding an AAV capsid protein (e.g., VP3) of thepresent invention.

In one specific embodiment, the surface-exposed lysine residuecorresponding to K532 of the wild-type AAV2 capsid sequence is modified.In one embodiment, the surface-exposed lysine residue of the AAV capsidis modified into glutamic acid (E) or arginine (R). In one specificembodiment, the surface-exposed lysine residue corresponding to K532 ofthe wild-type AAV2 capsid sequence is modified into arginine (K532R).

In one embodiment, at least one surface-exposed lysine residue of an AAVcapsid corresponding to a lysine position of a wild-type AAV2 capsidsequence is modified as indicated in FIG. 2B.

In one embodiment, at least one surface-exposed serine residuecorresponding to a serine residue selected from S662, S261, S458, S276,S658, S384, or S492 of the wild-type AAV capsid sequence (e.g., SEQ IDNOs: 1-10; in one embodiment, the capsid protein of wild-type AAV2 (SEQID NO:2)) is modified into a non-serine residue and/or is chemicallymodified so that said non-serine residue or said modified serine residuedoes not result in phosphorylation and/or ubiquitination of an AAVvector.

In one specific embodiment, the surface-exposed serine residuecorresponding S662 of the wild-type AAV2 capsid sequence is modified. Inone embodiment, the surface-exposed serine residue of the AAV capsid ismodified into valine (V), aspartic acid (D), or histidine (H). In onespecific embodiment, the surface-exposed serine residue corresponding toS662 of the wild-type AAV2 capsid sequence is modified into valine(S662V).

In one embodiment, a surface-exposed threonine residue corresponding toa threonine residue selected from T455, T491, T550, T659, or T671 of thewild-type AAV capsid sequence [e.g., SEQ ID NOs:1-10; and in aparticular embodiment, the capsid protein of wild-type AAV2 (SEQ IDNO:2)] is modified into a non-threonine residue and/or is chemicallymodified so that said non-threonine residue or said modified threonineresidue does not result in phosphorylation and/or ubiquitination of anAAV vector.

In one specific embodiment, the surface-exposed threonine residuecorresponding to T491 of the wild-type AAV2 capsid sequence is modified.In one embodiment, the surface-exposed threonine residue of the AAVcapsid is modified into valine (V). In one specific embodiment, thesurface-exposed threonine residue corresponding to T491 of the wild-typeAAV2 capsid sequence is modified into valine (T491V).

In one embodiment, the AAV vector comprises a modification ofsurface-exposed threonine residues at positions corresponding to(T550V+T659V+T491V) of the wild-type AAV capsid sequence [e.g., SEQ IDNOs:1-10; and in a particular embodiment, the capsid protein ofwild-type AAV2 (SEQ ID NO:2)]. In one embodiment, a surface-exposedtyrosine residue corresponding to a tyrosine residue selected from Y252,Y272, Y444, Y500, Y704, Y720, Y730, or Y673 of the wild-type AAV capsidsequence (e.g., SEQ ID NOs:1-10; in one embodiment, the capsid proteinof wild-type AAV2 (SEQ ID NO:2)) is modified into a non-tyrosine residueand/or is chemically modified so that said non-tyrosine residue or saidmodified tyrosine residue does not result in phosphorylation and/orubiquitination of an AAV vector.

In one embodiment, the surface-exposed tyrosine residue of the AAVcapsid is modified into phenylalanine (F). In one embodiment, the AAVvector comprises a modification of surface-exposed tyrosine residues atpositions corresponding to (Y730F+Y500F+Y444F) of the wild-type AAVcapsid sequence [e.g., SEQ ID NO:1 through SEQ ID NO:10; and in aparticular embodiment, the capsid protein of wild-type AAV2 (SEQ IDNO:2)].

In one embodiment, a combination of surface-exposed lysine, serine,threonine and/or tyrosine residues of the AAV capsid is modified,wherein the modification occurs at positions corresponding to(Y444F+Y500F+Y730F+T491V), (Y444F+Y500F+Y730F+T491V+T550V),(Y444F+Y500F+Y730F+T491V+T659V), (T491V+T550V+T659V),(Y440F+Y500F+Y730F), (Y444F+Y500F+Y730F+T491V+S662V), and/or(Y444F+Y500F+Y730F+T491V+T550V+T659V) of the wild-type AAV capsidsequence [e.g., SEQ ID NO:1 through SEQ ID NO:10; in one embodiment, thecapsid protein of wild-type AAV2 (SEQ ID NO:2)]. Also provided are AAVcapsid proteins encoded by the nucleic acid molecules of the presentinvention.

In one embodiment, the present invention provides a recombinantadeno-associated viral (rAAV) vector comprising a nucleic acid sequencethat encodes an AAV capsid protein of the invention. In anotherembodiment, the present invention provides a rAAV virion comprising anAAV capsid protein of the invention. In one embodiment, the rAAV vectorand virion has enhanced transduction efficiency, when compared to thewild-type rAAV vector and virion. In another embodiment, the rAAV vectorand virion is capable of efficient transduction of cells, tissues,and/or organs of interest.

In one embodiment, the rAAV vector further comprises a transgene (alsoreferred to as a heterologous nucleic acid molecule) operably linked toa promoter and optionally, other regulatory elements. In one embodiment,the transgene encodes a therapeutic agent of interest.

Exemplary promoters include one or more heterologous, tissue-specific,constitutive or inducible promoters, including, but not limited to, apromoter selected from the group consisting of cytomegalovirus (CMV)promoters, desmin (DES), beta-actin promoters, insulin promoters,enolase promoters, BDNF promoters, NGF promoters, EGF promoters, growthfactor promoters, axon-specific promoters, dendrite-specific promoters,brain-specific promoters, hippocampal-specific promoters,kidney-specific promoters, elafin promoters, cytokine promoters,interferon promoters, growth factor promoters, alpha-1 antitrypsinpromoters, brain-specific promoters, neural cell-specific promoters,central nervous system cell-specific promoters, peripheral nervoussystem cell-specific promoters, interleukin promoters, serpin promoters,hybrid CMV promoters, hybrid .beta.-actin promoters, EF1 promoters, U1apromoters, U1b promoters, Tet-inducible promoters and VP16-LexΛpromoters. In exemplary embodiments, the promoter is a mammalian oravian beta-actin promoter.

Exemplary enhancer sequences include, but are not limited to, one ormore selected from the group consisting of CMV enhancers, syntheticenhancers, liver-specific enhancers, vascular-specific enhancers,brain-specific enhancers, neural cell-specific enhancers, lung-specificenhancers, muscle-specific enhancers, kidney-specific enhancers,pancreas-specific enhancers, and islet cell-specific enhancers.

Exemplary therapeutic agents include, but are not limited to, an agentselected from the group consisting of polypeptides, peptides,antibodies, antigen binding fragments, ribozymes, peptide nucleic acids,siRNA, RNAi, antisense oligonucleotides and antisense polynucleotides.

In exemplary embodiments, the rAAV vectors of the invention will encodea therapeutic protein or polypeptide selected from the group consistingof adrenergic agonists, anti-apoptosis factors, apoptosis inhibitors,cytokine receptors, cytokines, cytotoxins, erythropoietic agents,glutamic acid decarboxylases, glycoproteins, growth factors, growthfactor receptors, hormones, hormone receptors, interferons,interleukins, interleukin receptors, kinases, kinase inhibitors, nervegrowth factors, netrins, neuroactive peptides, neuroactive peptidereceptors, neurogenic factors, neurogenic factor receptors, neuropilins,neurotrophic factors, neurotrophins, neurotrophin receptors,N-methyl-D-aspartate antagonists, plexins, proteases, proteaseinhibitors, protein decarboxylases, protein kinases, protein kinsaseinhibitors, protcolytic proteins, proteolytic protein inhibitors,semaphorin a semaphorin receptors, serotonin transport proteins,serotonin uptake inhibitors, serotonin receptors, serpins, serpinreceptors, and tumor suppressors.

In certain applications, the modified high-transduction efficiencyvectors may comprise a nucleic acid segment that encodes a polypeptideselected from the group consisting of BDNF, CNTF, CSF, EGF, FGF, G-SCF,GM-CSF, gonadotropin, IFN, IFG-1, M-CSF, NGF, PDGF, PEDF, TGF, TGF-B2,TNF, VEGF, prolactin, somatotropin, XIAP1, IL-1, IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-9, IL-10, IL-10(187A), viral IL-10, IL-11, IL-12,IL-13, IL-14, IL-15, IL-16, IL-17, and IL-18. Such therapeutic agentsmay be of human, murine, avian, porcine, bovine, ovine, feline, canine,equine, epine, caprine, lupine or primate origin.

Recombinant AAV vectors useful according to the invention includesingle-stranded (ss) or self-complementary (sc) AAV vectors.

The rAAV vectors of the present invention may also be within an isolatedmammalian host cell, including for example, human, primate, murine,feline, canine, porcine, ovine, bovine, equine, epine, caprine andlupine host cells. The rAAV vectors may be within an isolated mammalianhost cell such as a human endothelial, epithelial, vascular, liver,lung, heart, pancreas, intestinal, kidney, cardiac, cancer or tumor,muscle, bone, neural, blood, or brain cell.

Therapeutic Uses

Another aspect of the invention pertains to uses of the rAAV vectors andvirions of the invention for efficient transduction of cells, tissues,and/or organs of interest, and/or for use in gene therapy.

In one embodiment, the present invention provides a method fortransduction of cells, tissues, and/or organs of interest, comprisingintroducing into a cell, a composition comprising an effective amount ofa rAAV vector and/or virion of present invention.

In certain embodiments, rAAV vectors and virions of the invention areused for transduction of mammalian host cells, including for example,human, primate, murine, feline, canine, porcine, ovine, bovine, equine,epine, caprine and lupine host cells. In certain embodiments, the rAAVvectors and virions of the invention are used for transduction ofendothelial, epithelial, vascular, liver, lung, heart, pancreas,intestinal, kidney, muscle, bone, dendritic, cardiac, neural, blood,brain, fibroblast or cancer cells.

In one embodiment, cells, tissues, and/or organs of a subject aretransduced using the rAAV vectors and/or virions of the presentinvention.

The term “subject,” as used herein, describes an organism, includingmammals such as primates, to which treatment with the compositionsaccording to the present invention can be provided. Mammalian speciesthat can benefit from the disclosed methods of treatment include, butare not limited to, apes; chimpanzees; orangutans; humans; monkeys;domesticated animals such as dogs and cats; livestock such as horses,cattle, pigs, sheep, goats, and chickens; and other animals such asmice, rats, guinea pigs, and hamsters.

In addition, the present invention provides a method for treatment of adisease, wherein the method comprises administering, to a subject inneed of such treatment, an effective amount of a composition comprisingthe rAAV vector and/or virion of the invention.

The term “treatment” or any grammatical variation thereof (e.g., treat,treating, and treatment etc.), as used herein, includes but is notlimited to, alleviating a symptom of a disease or condition; and/orreducing, suppressing, inhibiting, lessening, ameliorating or affectingthe progression, severity, and/or scope of a disease or condition.

The term “effective amount,” as used herein, refers to an amount that iscapable of treating or ameliorating a disease or condition or otherwisecapable of producing an intended therapeutic effect.

The invention also provides for the use of a composition disclosedherein in the manufacture of a medicament for treating, preventing orameliorating the symptoms of a disease, disorder, dysfunction, injury ortrauma, including, but not limited to, the treatment, prevention, and/orprophylaxis of a disease, disorder or dysfunction, and/or theamelioration of one or more symptoms of such a disease, disorder ordysfunction.

Exemplary conditions for which rAAV viral based gene therapy may findparticular utility include, but are not limited to, cancer, diabetes,autoimmune disease, kidney disease, cardiovascular disease, pancreaticdisease, intestinal disease, liver disease, neurological disease,neuromuscular disorder, neuromotor deficit, neuroskeletal impairment,neurological disability, neurosensory dysfunction, stroke,.alpha..sub.1-antitrypsin (AAT) deficiency, Batten's disease, ischemia,an eating disorder, Alzheimer's disease, Huntington's disease,Parkinson's disease, skeletal disease and pulmonary disease.

The invention also provides a method for treating or ameliorating thesymptoms of such a disease, injury, disorder, or dysfunction in amammal. Such methods generally involve at least the step ofadministering to a mammal in need thereof, one or more of the rAAVvectors and virions of the present invention, in an amount and for atime sufficient to treat or ameliorate the symptoms of such a disease,injury, disorder, or dysfunction in the mammal.

Such treatment regimens are particularly contemplated in human therapy,via administration of one or more compositions either intramuscularly,intravenously, subcutaneously, intrathecally, intraperitoneally, or bydirect injection into an organ or a tissue of the subject under care.

The invention also provides a method for providing to a mammal in needthereof, a therapeutically-effective amount of the rAAV compositions ofthe present invention, in an amount, and for a time effective to providethe patient with a therapeutically-effective amount of the desiredtherapeutic agent(s) encoded by one or more nucleic acid segmentscomprised within the rAAV vector. Preferably, the therapeutic agent isselected from the group consisting of a polypeptide, a peptide, anantibody, an antigen binding fragment, a ribozyme, a peptide nucleicacid, a siRNA, an RNAi, an antisense oligonucleotide and an antisensepolynucleotide.

Pharmaceutical Compositions

The present invention also provides therapeutic or pharmaceuticalcompositions comprising the active ingredient in a form that can becombined with a therapeutically or pharmaceutically acceptable carrier.The genetic constructs of the present invention may be prepared in avariety of compositions, and may also be formulated in appropriatepharmaceutical vehicles for administration to human or animal subjects.

The rAAV molecules of the present invention and compositions comprisingthem provide new and useful therapeutics for the treatment, control, andamelioration of symptoms of a variety of disorders, and in particular,articular diseases, disorders, and dysfunctions, including for exampleosteoarthritis, rheumatoid arthritis, and related disorders.

The invention also provides compositions comprising one or more of thedisclosed rAAV vectors, expression systems, virions, viral particles; ormammalian cells. As described hereinbelow, such compositions may furthercomprise a pharmaceutical excipient, buffer, or diluent, and may beformulated for administration to an animal, and particularly a humanbeing. Such compositions may further optionally comprise a liposome, alipid, a lipid complex, a microsphere, a microparticle, a nanosphere, ora nanoparticle, or may be otherwise formulated for administration to thecells, tissues, organs, or body of a subject in need thereof. Suchcompositions may be formulated for use in a variety of therapies, suchas for example, in the amelioration, prevention, and/or treatment ofconditions such as peptide deficiency, polypeptide deficiency, peptideoverexpression, polypeptide overexpression, including for example,conditions which result in diseases or disorders such as cancers,tumors, or other malignant growths, neurological deficit dysfunction,autoimmune diseases, articular diseases, cardiac or pulmonary diseases,ischemia, stroke, cerebrovascular accidents, transient ischemic attacks(TIA); diabetes and/or other diseases of the pancreas; cardiocirculatorydisease or dysfunction (including, e.g., hypotension, hypertension,atherosclerosis, hypercholesterolemia, vascular damage or disease;neural diseases (including, e.g., Alzheimer's, Huntington's, Tay-Sach'sand Parkinson's disease, memory loss, trauma, motor impairment,neuropathy, and related disorders); biliary, renal or hepatic disease ordysfunction; musculoskeletal or neuromuscular diseases (including, e.g.,arthritis, palsy, cystic fibrosis (CF), amyotrophic lateral sclerosis(ALS), multiple sclerosis (MS), muscular dystrophy (MD), and such like).

In one embodiment, the number of rAAV vector and/or virion particlesadministered to a mammal may be on the order ranging from 10³ to 10¹³particles/ml, or any values therebetween, such as for example, about10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², or 10¹³ particles/ml. In oneembodiment, rAAV vector and/or virion particles of higher than 10¹³particles/ml are be administered. The rAAV vectors and/or virions can beadministered as a single dose, or divided into two or moreadministrations as may be required to achieve therapy of the particulardisease or disorder being treated. In most rAAV-based gene therapyregimens, the inventors believe that a lower titer of infectiousparticles will be required when using the modified-capsid rAAV vectors,than compared to conventional gene therapy protocols.

In certain embodiments, the present invention concerns formulation ofone or more rAAV-based compositions disclosed herein in pharmaceuticallyacceptable solutions for administration to a cell or an animal, eitheralone or in combination with one or more other modalities of therapy,and in particular, for therapy of human cells, tissues, and diseasesaffecting man.

If desired, nucleic acid segments, RNA, DNA or PNA compositions thatexpress one or more of therapeutic gene products may be administered incombination with other agents as well, such as, e.g., proteins orpolypeptides or various pharmaceutically-active agents, including one ormore systemic or topical administrations of therapeutic polypeptides,biologically active fragments, or variants thereof. In fact, there isvirtually no limit to other components that may also be included, giventhat the additional agents do not cause a significant adverse effectupon contact with the target cells or host tissues. The rAAV-basedgenetic compositions may thus be delivered along with various otheragents as required in the particular instance. Such compositions may bepurified from host cells or other biological sources, or alternativelymay be chemically synthesized as described herein. Likewise, suchcompositions may further comprise substituted or derivatized RNA, DNA,siRNA, mRNA, tRNA, ribozyme, catalytic RNA molecules, or PNAcompositions and such like.

Formulation of pharmaceutically-acceptable excipients and carriersolutions is well-known to those of skill in the art, as is thedevelopment of suitable dosing and treatment regimens for using theparticular compositions described herein in a variety of treatmentregimens, including e.g., oral, parenteral, intravenous, intranasal,intra-articular, intramuscular administration and formulation.

Typically, these formulations may contain at least about 0.1% of theactive compound or more, although the percentage of the activeingredient(s) may, of course, be varied and may conveniently be betweenabout 1 or 2% and about 70% or 80% or more of the weight or volume ofthe total formulation. Naturally, the amount of active compound(s) ineach therapeutically-useful composition may be prepared is such a waythat a suitable dosage will be obtained in any given unit dose of thecompound. Factors such as solubility, bioavailability, biologicalhalf-life, route of administration, product shelf life, as well as otherpharmacological considerations will be contemplated by one skilled inthe art of preparing such pharmaceutical formulations, and as such, avariety of dosages and treatment regimens may be desirable.

In certain circumstances it will be desirable to deliver the AAVvector-based therapeutic constructs in suitably formulatedpharmaceutical compositions disclosed herein either subcutaneously,intraocularly, intravitreally, parenterally, subcutaneously,intravenously, intracerebro-ventricularly, intramuscularly,intrathecally, orally, intraperitoneally, by oral or nasal inhalation,or by direct injection to one or more cells, tissues, or organs bydirect injection. The methods of administration may also include thosemodalities as described in U.S. Pat. Nos. 5,543,158, 5,641,515 and/or5,399,363 (each of which is specifically incorporated herein in itsentirety by express reference thereto). Solutions of the activecompounds as freebase or pharmacologically acceptable salts may beprepared in sterile water and may also suitably mixed with one or moresurfactants, such as hydroxypropylcellulose. Dispersions may also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms of the AAV-based viral compositions suitablefor injectable use include sterile aqueous solutions or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersions (U.S. Pat. No. 5,466,468, specificallyincorporated herein in its entirety by express reference thereto). Inall cases the form must be sterile and must be fluid to the extent thateasy syringability exists. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms, such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (e.g., glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), suitable mixtures thereof, and/or vegetable oils.Proper fluidity may be maintained, for example, by the use of a coating,such as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants.

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which the compound is administered. Such pharmaceutical carrierscan be sterile liquids, such as water and oils, including those ofpetroleum oil such as mineral oil, vegetable oil such as peanut oil,soybean oil, and sesame oil, animal oil, or oil of synthetic origin.Saline solutions and aqueous dextrose and glycerol solutions can also beemployed as liquid carriers.

The compositions of the present invention can be administered to thesubject being treated by standard routes including, but not limited to,pulmonary, intranasal, oral, inhalation, parenteral such as intravenous,topical, transdermal, intradermal, transmucosal, intraperitoneal,intramuscular, intracapsular, intraorbital, intracardiac, transtracheal,subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid,intraspinal, epidural and intrasternal injection. In preferredembodiments, the composition is administered via intranasal, pulmonary,or oral route.

For administration of an injectable aqueous solution, for example, thesolution may be suitably buffered, if necessary, and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, a sterile aqueous medium that can be employed will be knownto those of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, and the general safety and purity standards as required byFDA Office of Biologics standards.

Sterile injectable solutions are prepared by incorporating the activeAAV vector-delivered therapeutic polypeptide-encoding DNA fragments inthe required amount in the appropriate solvent with several of the otheringredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thevarious sterilized active ingredients into a sterile vehicle whichcontains the basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum-drying and freeze-drying techniques which yield apowder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof.

The AAV vector compositions disclosed herein may also be formulated in aneutral or salt form. Pharmaceutically-acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike. Upon formulation, solutions will be administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective. The formulations are easily administered in avariety of dosage forms such as injectable solutions, drug-releasecapsules, and the like.

The amount of AAV compositions and time of administration of suchcompositions will be within the purview of the skilled artisan havingbenefit of the present teachings. It is likely, however, that theadministration of therapeutically-effective amounts of the disclosedcompositions may be achieved by a single administration, such as forexample, a single injection of sufficient numbers of infectiousparticles to provide therapeutic benefit to the patient undergoing suchtreatment. Alternatively, in some circumstances, it may be desirable toprovide multiple, or successive administrations of the AAV vectorcompositions, either over a relatively short, or a relatively prolongedperiod of time, as may be determined by the medical practitioneroverseeing the administration of such compositions.

Expression Vectors

The present invention contemplates a variety of AAV-based expressionsystems, and vectors. In one embodiment the preferred AAV expressionvectors comprise at least a first nucleic acid segment that encodes atherapeutic peptide, protein, or polypeptide. In another embodiment, thepreferred AAV expression vectors disclosed herein comprise at least afirst nucleic acid segment that encodes an antisense molecule. Inanother embodiment, a promoter is operatively linked to a sequenceregion that encodes a functional mRNA, a tRNA, a ribozyme or anantisense RNA.

The choice of which expression vector and ultimately to which promoter apolypeptide coding region is operatively linked depends directly on thefunctional properties desired, e.g., the location and timing of proteinexpression, and the host cell to be transformed. These are well knownlimitations inherent in the art of constructing recombinant DNAmolecules. However, a vector useful in practicing the present inventionis capable of directing the expression of the functional RNA to which itis operatively linked.

A variety of methods have been developed to operatively link DNA tovectors via complementary cohesive termini or blunt ends. For instance,complementary homopolymer tracts can be added to the DNA segment to beinserted and to the vector DNA. The vector and DNA segment are thenjoined by hydrogen bonding between the complementary homopolymeric tailsto form recombinant DNA molecules.

To express a therapeutic agent in accordance with the present inventionone may prepare a tyrosine-modified rAAV expression vector thatcomprises a therapeutic agent-encoding nucleic acid segment under thecontrol of one or more promoters. To bring a sequence “under the controlof” a promoter, one positions the 5′ end of the transcription initiationsite of the transcriptional reading frame generally between about 1 andabout 50 nucleotides “downstream” of (i.e., 3′ of) the chosen promoter.The “upstream” promoter stimulates transcription of the DNA and promotesexpression of the encoded polypeptide. This is the meaning of“recombinant expression” in this context. Particularly preferredrecombinant vector constructs are those that comprise a rAAV vector.Such vectors are described in detail herein.

When the use of such vectors is contemplated for introduction of one ormore exogenous proteins, polypeptides, peptides, ribozymes, and/orantisense oligonucleotides, to a particular cell transfected with thevector, one may employ the rAAV vectors or the tyrosine-modified rAAVvectors disclosed herein by genetically modifying the vectors to furthercomprise at least a first exogenous polynucleotide operably positioneddownstream and under the control of at least a first heterologouspromoter that expresses the polynucleotide in a cell comprising thevector to produce the encoded peptide, protein, polypeptide, ribozyme,siRNA, RNAi or antisense oligonucleotide. Such constructs may employheterologous promoters that are constitutive, inducible, or evencell-specific promoters. Exemplary such promoters include, but are notlimited to, viral, mammalian, and avian promoters, including for examplea CMV promoter, a .beta.-actin promoter, a hybrid CMV promoter, a hybrid.beta.-actin promoter, an EF1 promoter, a U1a promoter, a U1b promoter,a Tet-inducible promoter, a VP16-LexA promoter, and such like.

The vectors or expression systems may also further comprise one or moreenhancers, regulatory elements, transcriptional elements, to alter oreffect transcription of the heterologous gene cloned in the rAAVvectors. For example, the rAAV vectors of the present invention mayfurther comprise at least a first CMV enhancer, a synthetic enhancer, ora cell- or tissue-specific enhancer. The exogenous polynucleotide mayalso further comprise one or more intron sequences.

Therapeutic Kits

The invention also encompasses one or more of the genetically-modifiedrAAV vector compositions described herein together with one or morepharmaceutically-acceptable excipients, carriers, diluents, adjuvants,and/or other components, as may be employed in the formulation ofparticular rAAV-polynucleotide delivery formulations, and in thepreparation of therapeutic agents for administration to a subject, andin particularly, to a human. In particular, such kits may comprise oneor more of the disclosed rAAV compositions in combination withinstructions for using the viral vector in the treatment of suchdisorders in a subject, and may typically further include containersprepared for convenient commercial packaging.

As such, preferred animals for administration of the pharmaceuticalcompositions disclosed herein include mammals, and particularly humans.Other preferred animals include murines, bovines, equines, porcines,canines, and felines. The composition may include partially orsignificantly purified rAAV compositions, either alone, or incombination with one or more additional active ingredients, which may beobtained from natural or recombinant sources, or which may be obtainablenaturally or either chemically synthesized, or alternatively produced invitro from recombinant host cells expressing DNA segments encoding suchadditional active ingredients.

Therapeutic kits may also be prepared that comprise at least one of thecompositions disclosed herein and instructions for using the compositionas a therapeutic agent. The container means for such kits may typicallycomprise at least one vial, test tube, flask, bottle, syringe or othercontainer means, into which the disclosed rAAV composition(s) may beplaced, and preferably suitably aliquoted. Where a second therapeuticpolypeptide composition is also provided, the kit may also contain asecond distinct container means into which this second composition maybe placed. Alternatively, the plurality of therapeutic biologicallyactive compositions may be prepared in a single pharmaceuticalcomposition, and may be packaged in a single container means, such as avial, flask, syringe, bottle, or other suitable single container means.The kits of the present invention will also typically include a meansfor containing the vial(s) in close confinement for commercial sale,such as, e.g., injection or blow-molded plastic containers into whichthe desired vial(s) are retained.

AAV Capsid Proteins

Supramolecular assembly of 60 individual capsid protein subunits into anon-enveloped, T-1 icosahedral lattice capable of protecting a 4.7-kbsingle-stranded DNA genome is a critical step in the life-cycle of thehelper-dependent human parvovirus, adeno-associated virus 2 (AAV2). Themature 20-nm diameter AAV2 particle is composed of three structuralproteins designated VP1, VP2, and VP3 (molecular masses of 87, 73, and62 kDa respectively) in a ratio of 1:1:18. Based on its symmetry andthese molecular weight estimates, of the 60 capsid proteins comprisingthe particle, three are VP1 proteins, three are VP2 proteins, andfifty-four are VP3 proteins.

Biological Functional Equivalents

Modification and changes to the structure of the polynucleotides andpolypeptides of wild-type rAAV vectors to provide the improved rAAVvirions as described in the present invention to obtain functional viralvectors that possess desirable characteristics, particularly withrespect to improved delivery of therapeutic gene constructs to selectedmammalian cell, tissues, and organs for the treatment, prevention, andprophylaxis of various diseases and disorders, as well as means for theamelioration of symptoms of such diseases, and to facilitate theexpression of exogenous therapeutic and/or prophylactic polypeptides ofinterest via rAAV vector-mediated gene therapy. As mentioned above, oneof the key aspects of the present invention is the creation of one ormore mutations into specific polynucleotide sequences that encode one ormore of the therapeutic agents encoded by the disclosed rAAV constructs.In certain circumstances, the resulting polypeptide sequence is alteredby these mutations, or in other cases, the sequence of the polypeptideis unchanged by one or more mutations in the encoding polynucleotide toproduce modified vectors with improved properties for effecting genetherapy in mammalian systems.

For example, certain amino acids may be substituted for other aminoacids in a protein structure without appreciable loss of interactivebinding capacity with structures such as, for example, antigen-bindingregions of antibodies or binding sites on substrate molecules. Since itis the interactive capacity and nature of a protein that defines thatprotein's biological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence, and, of course, itsunderlying DNA coding sequence, and nevertheless obtain a protein withlike properties. It is thus contemplated by the inventors that variouschanges may be made in the polynucleotide sequences disclosed herein,without appreciable loss of their biological utility or activity.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982, incorporate herein byreference). It is accepted that the relative hydropathic character ofthe amino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like. Each amino acid has been assigned a hydropathicindex on the basis of their hydrophobicity and charge characteristics(Kyte and Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2);leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

Exemplary Definitions

In accordance with the present invention, polynucleotides, nucleic acidsegments, nucleic acid sequences, and the like, include, but are notlimited to, DNAs (including and not limited to genomic or extragenomicDNAs), genes, peptide nucleic acids (PNAs) RNAs (including, but notlimited to, rRNAs, mRNAs and tRNAs), nucleosides, and suitable nucleicacid segments either obtained from natural sources, chemicallysynthesized, modified, or otherwise prepared or synthesized in whole orin part by the hand of man.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andcompositions similar or equivalent to those described herein can be usedin the practice or testing of the present invention, the preferredmethods and compositions are described herein. For purposes of thepresent invention, the following terms are defined below:

The term “promoter,” as used herein refers to a region or regions of anucleic acid sequence that regulates transcription.

The term “regulatory element,” as used herein, refers to a region orregions of a nucleic acid sequence that regulates transcription.Exemplary regulatory elements include, but are not limited to,enhancers, post-transcriptional elements, transcriptional controlsequences, and such like.

The term “vector,” as used herein, refers to a nucleic acid molecule(typically comprised of DNA) capable of replication in a host celland/or to which another nucleic acid segment can be operatively linkedso as to bring about replication of the attached segment. A plasmid,cosmid, or a virus is an exemplary vector.

The term “substantially corresponds to,” “substantially homologous,” or“substantial identity,” as used herein, denote a characteristic of anucleic acid or an amino acid sequence, wherein a selected nucleic acidor amino acid sequence has at least about 70 or about 75 percentsequence identity as compared to a selected reference nucleic acid oramino acid sequence. More typically, the selected sequence and thereference sequence will have at least about 76, 77, 78, 79, 80, 81, 82,83, 84 or even 85 percent sequence identity, and more preferably atleast about 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 percent sequenceidentity. More preferably still, highly homologous sequences often sharegreater than at least about 96, 97, 98, or 99 percent sequence identitybetween the selected sequence and the reference sequence to which it wascompared.

The percentage of sequence identity may be calculated over the entirelength of the sequences to be compared, or may be calculated byexcluding small deletions or additions which total less than about 25percent or so of the chosen reference sequence. The reference sequencemay be a subset of a larger sequence, such as a portion of a gene orflanking sequence, or a repetitive portion of a chromosome. However, inthe case of sequence homology of two or more polynucleotide sequences,the reference sequence will typically comprise at least about 18-25nucleotides, more typically at least about 26 to 35 nucleotides, andeven more typically at least about 40, 50, 60, 70, 80, 90, or even 100or so nucleotides.

Desirably, which highly homologous fragments are desired, the extent ofpercent identity between the two sequences will be at least about 80%,preferably at least about 85%, and more preferably about 90% or 95% orhigher, as readily determined by one or more of the sequence comparisonalgorithms well-known to those of skill in the art, such as e.g., theFASTA program analysis described by Pearson and Lipman (Proc. Natl.Acad. Sci. USA, 85(8):2444-8, April 1988).

The term “operably linked,” as used herein, refers to that the nucleicacid sequences being linked are typically contiguous, or substantiallycontiguous, and, where necessary to join two protein coding regions,contiguous and in reading frame. However, since enhancers generallyfunction when separated from the promoter by several kilobases andintronic sequences may be of variable lengths, some polynucleotideelements may be operably linked but not contiguous.

EXAMPLES

Following are examples that illustrate procedures and embodiments forpracticing the invention. The examples should not be construed aslimiting.

Materials and Methods:

Cells and antibodies: HEK293, HeLa, NIH3T3 cells were obtained from theAmerican Type Culture Collection (Manassas, Va., USA) and maintained asmonolayer cultures in DMEM (Invitrogen) supplemented with 10% FBS(Sigma-Aldrich, St. Louis, Mo., USA) and antibiotics (Lonza).Leukapheresis-derived peripheral blood mononuclear cells (PBMCs)(AllCells) were purified on Ficoll-Paque (GEHeathCare), re-suspended inserum-free AIM-V medium (Lonza), and semi-adherent cell fractions wereincubated for 7 days with recombinant human IL-4 (500 U/mL) and GM-CSF(800 U/mL) (R&D Systems). Cell maturation was initiated with cytokinemixture including 10 ng/mL TNF-α, 10 ng/mL IL-1, 10 ng/mL IL-6, and 1mg/mL PGE2 (R&D Systems) for 48 hrs. Prior to EGFP expression, cellswere characterized for co-stimulatory molecules expression to ensurethat they met the typical phenotype of mature dendritic cells (mDC)(CD80, RPE, murine IgG1; CD83, RPE, murine IgG1; CD86, FITC, murineIgG1; Invitrogen).

Site-directed mutagenesis: A two-stage PCR was performed with plasmidpACG2 as described previously (Wang et al., 1999) using Turbo® PfuPolymerase (Stratagene). Briefly, in stage one, two PCR extensionreactions were performed in separate tubes for the forward and reversePCR primer for 3 cycles. In stage two, the two reactions were mixed anda PCR reaction was performed for an additional 15 cycles, followed byDpnI digestion for one hr. Primers were designed to introduce changesfrom serine (TCA or AGC) to valine (GTA or GTC) for each of the residuesmutated.

Production of recombinant AAV vectors: Recombinant AAV2 vectorscontaining the EGFP gene driven by the chicken ®-actin promoter weregenerated as described previously (Zolotukhin, 2002). Briefly, HEK293cells were transfected using Polyethelenimine (PEI, linear, MW 25,000,Polysciences, Inc.). Seventy-two hrs' post transfection, cells wereharvested and vectors were purified by iodixanol (Sigma-Aldrich)gradient centrifugation and ion exchange column chromatography (HiTrapSp Hp 5 mL, GE Healthcare). Virus was then concentrated and the bufferwas exchanged in three cycles to lactated Ringer's using centrifugalspin concentrators (Apollo, 150-kDa cut-off 20-mL capacity, CLP) (Chenget al., 2011). Ten μL of purified virus was treated with DNAse I(Invitrogen) for 2 hrs at 37° C., then additional 2 hrs with proteinaseK (Invitrogen) at 56° C. The reaction mixture was purified byphenol/chloroform, followed by chloroform treatment. Packaged DNA wasprecipitated with ethanol in the presence of 20 μg glycogen(Invitrogen). DNAse I-resistant AAV particle titers were determined byRT-PCR with the following primers-pair, specific for the CBA promoter:

forward (SEQ ID NO: 11) 5′-TCCCATAGTAACGCCAATAGG-3′ reverse(SEQ ID NO: 12) 5′-CTTGGCATATGATACACTTGATG-3′

and SYBR Green PCR Master Mix (Invitrogen).

Recombinant AAV vector transduction assays In vitro: HEK293 ormonocyte-derived dendritic cells (moDCs), were transduced with AAV2vectors with 1,000 vgs/cell or 2,000 vgs/cell respectively, andincubated for 48 hrs. Alternatively, cells were pretreated with 50 μM ofselective serine/threonine kinases inhibitors2-(2-hydroxyethylamino)-6-aminohexylcarbamic acid tert-butylester-9-isopropylpurine (for CaMK-II), anthra[1,9-cd]pyrazol-6(2H)-one,1,9-pyrazoloanthrone (for JNK), and4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole(for MAPK) (CK59, JNK inhibitor 2, PD 98059, Calbiochem), 1 hr beforetransduction. Transgene expression was assessed as the total area ofgreen fluorescence (pixel²) per visual field (mean±SD) or by flowcytometry as described previously (Markusic et al., 2011; Jayandharan etal., 2011). Analysis of variance was used to compare test results andthe control, which were determined to be statistically significant.

Western blot analysis: Western blot analysis was performed as describedpreviously (Aslanidi et al., 2007). Cells were harvested bycentrifugation, washed with PBS, and resuspended in lysis buffercontaining 50 mM Tris_HCl, pH 7.5, 120 mM NaCl, 1% Nonidet P-40, 10%glycerol, 10 mM Na₄P₂O₇, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mMEDTA, and 1 mM EGTA supplemented with protease and phosphataseinhibitors mixture (Set 2 and 3, Calbiochem). The suspension wasincubated on ice for 1 hr and clarified by centrifugation for 30 min at14,000 rpm at 4° C. Following normalization for protein concentration,samples were separated using 12% polyacrylamide/SDS electrophoresis,transferred to a nitrocellulose membrane, and probed with primaryantibodies, anti p-p38 MAPK (Thr180/Tyr182) rabbit mAb, total p38 MAPKrabbit mAb and GAPDH rabbit mAb (1:1000, CellSignaling), follow bysecondary horseradish peroxidase-linked linked antibodies (1:1000,CellSignaling).

Specific cytotoxic T-lymphocytes generation and cytotoxicity assay:Monocytes-derived dendritic cells (moDCs) were generated as describedabove. Immature DCs were infected with AAV2-S662V vectors encoding humantelomerase cDNA (Dr. Karina Krotova, University of Florida), separatedinto two overlapping ORF—hTERT₈₃₈₋₂₂₂₉ and hTERT₂₀₄₂₋₃₄₅₄ at MOI 2,000vgs/cell of each. Cells were then allowed to undergo stimulation withsupplements to induce maturation. After 48 hr, the mature DCs expressinghTERT were harvested and mixed with the PBMCs at a ratio of 20:1. CTLswere cultured in AIM-V medium containing recombinant human IL-15 (20IU/ml) and IL-7 (20 ng/mL) at 20×10⁶ cells in 25 cm² flasks. Freshcytokines were added every 2 days. After 7 days post-priming, the cellswere harvested and used for killing assays (Heiser et al., 2002). Akilling curve was generated and specific cell lysis was determined byFACS analysis of live/dead cell ratios as described previously (Mattiset al., 1997). Human immortalized myelogenous leukemia cell line, K562,was used as a target.

Statistical analysis: Results were presented as mean±S.D. Differencesbetween groups were identified using a grouped-unpaired two-taileddistribution of Student's t-test. P-values <0.05 were consideredstatistically significant.

Example 1—Inhibition of Specific Cellular Serine/Threonine KinaseIncreases Transduction Efficiency of rAAV2 Vectors

Inhibition of cellular epidermal growth factor receptor protein tyrosinekinase (EGFR-PTK) activity, as well as site-directed mutagenesis of theseven surface-exposed tyrosine residues significantly increased thetransduction efficiency of AAV2 vectors by preventing phosphorylation ofthese residues, thereby circumventing ubiquitination and subsequentproteasome-mediated degradation of the vectors. AAV2 capsids alsocontain fifteen surface-exposed serine residues, which can potentiallybe phosphorylated by cellular serine/threonine kinases widely expressedin various cell types and tissues.

To examine whether inhibition of such kinase activity can preventphosphorylation of surface-exposed serine residues, and thus, improveintracellular trafficking and nuclear transport of AAV2 vectors, severalcommercially available specific inhibitors of cellular serine/threoninekinases, such as calmodulin-dependent protein kinase II (CamK-II), c-JunN-terminal kinase (JNK), and mitogen-activated protein kinase (p38MAPK), were used. HEK293 cells were pre-treated with specificinhibitors, such as 2-(2-hydroxyethylamino)-6-aminohexylcarbamic acidtert-butyl ester-9-isopropylpurine (for CaMK-II),anthra[1,9-cd]pyrazol-6(2H)-one, 1,9-pyrazoloanthrone (for JNK), and4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole(for p38 MAPK) for 1 hr at various concentrations. Cells weresubsequently transduced with either single-stranded (ss) orself-complementary (sc) AAV2 vectors at 1,000 vector genomes (vgs) percell.

These results show that all inhibitors at a concentration of 50 μMsignificantly increased the transduction efficiency of ssAAV2 and scAAV2vectors, the p38 MAPK inhibitor being the most effective (FIG. 3A andFIG. 3B). The results indicate that the increase in the transductionefficiency was due to prevention of phosphorylation of vector capsidsrather than improved viral second-strand DNA synthesis.

Example 2—Site-Directed Mutagenesis of Surface-Exposed Serine ResiduesImproves Vector-Mediated Transgene Expression

The AAV2 capsid contains 50 serine (S) residues in the viral protein 3(VP3) common region of the three capsid VPs, of which 15 residues (S261,S267, S276, S384, S468, S492, S498, S578, S658, S662, S668, S707, S721)are surface-exposed. Each of the 15 S residues was substituted withvaline (V) residues by site-directed mutagenesis as describedpreviously. Most mutants could be generated at titers similar to the WTAAV2 vectors, with the exception of S261V, S276V, and S658V, which wereproduced at ˜10 times lower titers, and S267V and S668V, which producedno detectable levels of DNAse I-resistant vector particles. The titersof S468V and S384V mutants were ˜3-5 times higher than the WT AAV2vectors. Each of the S-V mutant vectors was evaluated for transductionefficiency in 293 cells.

These results, shown in FIG. 4A and FIG. 4B, indicated that of the 15mutants, the S662V mutant transduced 293 cells ˜20-fold more efficientlythan its WT counterpart. The transduction efficiency of the S458V andthe S492V mutant vectors was increased by ˜4- and 2-fold, respectively.The transduction efficiency of the S468V and the S384V mutants, whichwere produced at titers higher than the WT AAV2 vectors, either remainedunchanged (S468V), or reduced ˜10-fold (S384V) at the same multiplicityof infection (MOI). The transduction efficiency of variousserine-to-valine mutated AAV2 vectors is summarized in FIG. 5.Surprisingly, no further increase in transduction efficiency wasobserved in vectors containing either of the double-mutants (S458V+S662Vand S492V+S662V), or in a vector that contained the triple-mutant(S458V+S492V+S662V).

Example 3—Substitution of S662 with Various Amino Acids

In addition to the S-to-V substitution at position 662, the followingseven mutants were also generated with different amino acidsubstitutions: S662→Alanine (A), S662→Asparagine (N), S662→Aspartic acid(D), S662→Histidine (H), S662→Isoleucine (I), S662→Leucine (L), andS662→Phenylalanine (F). The transduction efficiency of each of thesemutant vectors was also evaluated in 293 cells similar to that asdescribed above.

The results, as shown in FIG. 6A and FIG. 6B, and summarized in FIG. 7,demonstrate that the substitution of S with V led to production of themost efficient mutant without any change in vector titers, when comparedto other mutants. Replacement of S with N, I, L, or F decreased thepackaging efficiency ˜10-fold with no significant effect on thetransduction efficiency, whereas substitution with D or H increased thetransduction efficiency ˜8-fold and ˜4-fold, respectively, with noeffect on vector titers. Substitution of S to A increased the viraltiter up to ˜5-fold, and enhanced the transgene expression ˜3-foldcompared with the WT AAV2 vector. The observed variability in titers andinfectivity of the serine-mutants at position 662 suggests the criticalrole each of the amino acids plays in modulating both the AAV2 packagingefficiency and its biological activity.

Example 4—Transduction Efficiency of S662V Vectors Correlated with P38MAPK Activity in Various Cell Types

The S662V vector-mediated transgene expression is examined using thefollowing cells types: (i) NIH3T3 (mouse embryonic fibroblasts), (ii)H2.35 (mouse fetal hepatocytes), (iii) HeLa (human cervical cancercells), and (iv) primary human monocyte-derived dendritic cells (moDCs).These cell types were transduced with WT scAAV2-EGFP or S662VscAAV2-EGFP vectors at an MOI of 2,000 vgs per cell under identicalconditions. EGFP gene expression was evaluated 48 hrs post-infection(p.i.) for HeLa, 293 and moDCs, and 5 days p.i. for H2.35 and NIH3T3cells.

The results, as shown in FIG. 8A, show that although the absolutedifferences in the transduction efficiency between WT and S662V mutantvectors ranged from ˜3-fold (in H2.35 cells) to ˜20-fold (in 293 cells),the mutant vector was consistently more efficient in each cell typetested.

To examine whether the observed differences in the transductionefficiency of the WT and the mutant vectors is due to variations in thelevels of expression and/or activity of the cellular p38 MAPK, celllysates prepared from each cell type were analyzed on Western blotsprobed with specific antibodies to detect both total p38 MAPK andphospho-p38 MAPK levels. GAPDH was used as a loading control.

The results, as shown in FIG. 8B, indicate that while the p38 MAPKprotein levels were similar, the kinase activity, as determined by thelevel of phosphorylation, varied significantly among different celltypes, and the transduction efficiency of the S662V mutant vectorcorrelated roughly with the p38 MAPK activity. The results show p38MAPK-mediated phosphorylation of AAV2 vectors. In addition, transductionby the WT-AAV2 vectors did not lead to up-regulation of phosphorylationof p38 MAPK in either 293 cells or in moDCs; this indicates that AAVdoes not induce robust phenotypic changes in moDCs.

Example 5—S662V Mutant Vector-Mediated Transduction of moDCs Did notLead to Phenotypic Alterations

MAPK family members play important roles in the development andmaturation of APCs. In this Example, moDCs, isolated from healthy donorleukapheresis, were treated with 50 μM selective kinase inhibitors asdescribed above and then transduced with WT scAAV2-EGFP vectors. Two hrsp.i., cells were treated with supplements (TNF-α, IL-1β, Il-6, PGE2) toinduce maturation. EGFP transgene expression was evaluated 48 hrs p.i.by fluorescence microscopy.

The results show that pre-treatment of moDCs with specific inhibitors ofJNK and p38 MAPK increased EGFP expression levels ˜2-fold and ˜3-fold,respectively, and the transduction efficiency was enhanced by ˜5-foldwith the S662V mutant vectors (FIG. 9A, FIG. 9B, and FIG. 9C).

Since inhibition of these kinases has previously been reported toprevent maturation of dendritic cells, the capability of S662V mutant toinduce phenotypic changes in DCs was also examined. Briefly, moDC wereinfected with increasingly higher MOI of up to 50,000 vgs per cell,harvested at 48 hrs p.i., and analyzed by fluorescence-activated cellsorting (FACS) for up regulation of surface co-stimulatory molecules.Flow cytometric analyses of DC maturation markers such as CD80, CD83 andCD86 indicated that, similar to WT AAV2 vectors, the S662V mutantvectors also did not induce the maturation of moDCs (FIG. 9C). Theresults showed that the capsid-mutated AAV vectors prepared inaccordance with the present invention demonstrated low immunogenicity.

Example 6—Generation of hTERT-Specific CTL by moDC Transduced withAAV2-S662V Vectors

As the serine-mutant AAV2 vector-mediated transgene expression in moDCwas significantly improved compared with the WT-AAV2 vectors, this studydemonstrates the ability of S662V-loaded moDCs to stimulate thegeneration of cytotoxic T-lymphocytes and effective specific killing oftarget cells. Given that human telomerase is recognized as a uniqueanti-cancer target commonly expressed in most cancer cells, a truncatedhuman telomerase (hTERT) gene under the control of the chicken β-actinpromoter was cloned and the DNA was packaged into the AAV2 S662V mutant.Non-adherent peripheral blood mononuclear cells (PBMC) containing up to25% of CD8 positive cells were stimulated once with moDC/hTERT deliveredby the S662V vector. An immortalized myelogenous leukemia cell line,K562, was used for a two-color fluorescence assay of cell-mediatedcytotoxicity to generate a killing curve with subsequently reducedeffector to target cell ratio.

The results, shown in FIG. 10, indicated that moDC loaded with hTERTcould effectively stimulate specific T cell clone proliferation andkilling activity compared with moDC expressing GFP. These results alsoindicated that capsid-mutated rAAV-vector based delivery methods couldalso be used in a variety of mammalian vaccination methodologies.

Example 7—High-Efficiency rAAV2 Vectors Obtained by Site-DirectedMutagenesis of Surface-Exposed Tyrosine, Serine, and/or ThreonineResidues

AAV vectors are currently in use in a number of clinical trials as adelivery vehicle to target a variety of tissues to achieve sustainedexpression of therapeutic genes. However, large vector doses are neededto observe therapeutic benefits. Production of sufficient amounts of thevector also poses a challenge, as well as the risk of initiating animmune response to the vector. Thus, it is critical to develop novel AAVvectors with high transduction efficiency at lower doses.

The cellular epidermal growth factor receptor protein tyrosine kinase(EGFR-PTK) negatively impacts transgene expression from recombinant AAV2vectors primarily due to phosphorylation of AAV2 capsids at tyrosineresidues. Tyrosine-phosphorylated capsids are subsequently degraded bythe host proteasome machinery, which negatively impacts the transductionefficiency of AAV vectors. Selective inhibitors of JNK and p38 MAPKserine/threonine kinases improve the transduction efficiency, indicatingthat phosphorylation of certain surface-exposed serine or/and threonineresidues decreases the transduction efficiency of AAV vectors.

Site-directed mutagenesis to the capsid protein of the wild-type AAV2was performed. As shown in FIG. 11A, FIG. 11B, FIG. 12A, and FIG. 12B,the serine (S662V) and threonine (T491V) mutants of the wild-type AAV2capsid protein substantially increase the transduction efficiency of AAVvectors.

The serine (S662V) and threonine (T491V) mutations were combined withthe best-performing single (Y730F) and triple (Y730F+500+444F)tyrosine-mutants to generate the following vectors: (i) three double(S662V+T491V; Y730F+S662V; Y730F+T491V); (ii) one triple(S662V+Y730F+T491V); (iii) two quadruple (Y730+500+444F+S662V;Y730+500+44F+T491V); and (iv) one quintuple(Y730+500+4440F+S662V+T491V). The transduction efficiency of each of themutant vector was evaluated using a primary murine hepatocyte cell lineH3.25.

As shown in FIG. 13A and FIG. 13B, the AAV2 quadruple mutant(Y730+500+730F+T491V)-based vector increased the transduction efficiencyby approximately 30-fold over that of the corresponding, unmodifiedwild-type (WT) AAV2 vector, and approximately 3-fold over that producedby the AAV2 triple mutant (Y730+500+444F)-based vector. Combining theS662V mutation with either the single (Y730F)- or the triple-tyrosinemutant (Y730F+500+444F) vector, negatively affected the transductionefficiency.

Genetically modified dendritic cells (DCs) have been extensivelystudied, and numerous Phase I and II clinical trials evaluating theirefficacy in patients with cancer have been initiated. However, currentmethods for DC loading are inadequate in terms of cell viability,uncertainty regarding the longevity of antigen presentation, and therestriction by the patient's haplotype. Successful transduction ofdifferent subsets of DCs by different commonly used serotypes of AAVvectors has been demonstrated and the potential advantage of anAAV-based antitumor vaccine discussed. However, further improvements ingene transfer by recombinant AAV vectors to DCs in terms of specificityand transduction efficiency are warranted to achieve a significantimpact when used as an antitumor vaccine.

Serine/threonine protein kinases can negatively regulate the efficiencyof recombinant AAV vector-mediated transgene expression byphosphorylating the surface-exposed serine and/or threonine residues onthe viral capsid and target the vectors for proteasome-mediateddegradation. Prevention of phosphorylation of the surface-exposed serineand threonine residues could allow the vectors to evade phosphorylationand subsequent ubiquitination and, thus, prevent proteasomaldegradation.

Site-directed mutagenesis was performed to the wild-type AAV vector ofeach of the 15 surface-exposed serine (S) residues. The results showthat substitution of S662 to valine (V) increased the transductionefficiency of the S662V mutant up to 6-fold, when compared to thewild-type AAV2 vector. In addition, site-directed mutagenesis wasperformed to substitute each of the 17 surface-exposed threonine (T)residues of the wild-type AAV2 vector with V (T251V, T329V, T330V,T454V, T455V, T503V, T550V, T592V, T581V, T597V, T491V, T671V, T659V,T660V, T701V, T713V, T716V). The transduction efficiency of each of theT-V mutant vectors was evaluated using primary human monocyte-deriveddendritic cells (moDCs) at an MOI of 2,000 vgs/cell. Followingmaturation with a cytokine mixture including 10 ng/ml. TNF-α, 10 ng/mLIL-1, 10 ng/mL IL-6, and 1 mg/mL PGE2, EGFP expression was analyzed48-hrs' post-infection under a fluorescent microscope. Cells werecharacterized for expression of co-stimulatory molecules (CD80, CD83,and CD86) to ensure that they met the typical phenotype of maturedendritic cells (mDCs).

As shown in FIG. 14A and FIG. 14B, mutations of the following T residues(T455V, T491V, T550V, T659V, T671V) increased the transductionefficiency of moDCs up to 5-fold, with the T491V mutant demonstratingthe highest transduction efficiency among those tested in this study.

To examine whether multiple mutations of T residues could furtherenhance the transduction efficiency, the following AAV2 mutants werealso generated: (i) four AAV2 vectors with double mutations with respectto the wild-type AAV2 vector (T455V+T491V; T550V+T491V; T659V+T491V;T671V+T491V); (ii) two triple-mutated AAV2 vectors (T455V+T491V+T550Vand T550V+T659V+T491V); and (iii) one quadruple mutated AAV2 vector(T455V+T550V+T659V+T491V). The results demonstrated that several ofthese multiple-mutant vectors increased the transduction efficiency ofdendritic cells, and the triple-mutant (T550V+T659V+T491V) in particularwas shown to possess optimal transduction efficiency (approximatelyten-fold greater than that of the corresponding unmodified, wild-typeAAV2 vector!) These results were further enhanced by making variouscombinatorial mutants in “mix-and-match” fashion to generate a number ofsuitable capsid-mutated vectors/One such combination—combining the bestperforming serine substitution (S662V) mutant with the best-performingthreonine substitution (T491V) further enhanced the transductionefficiency by approximately 8-fold as compared to either of theindividual single mutations.

Example 8—Targeted Mutagenesis of Ubiquitin-Binding Lysine Residues onthe AAV2 Capsid Improves its Transduction Efficiency

It is now well recognized that hepatic gene transfer of high doses ofAAV vectors predispose to a robust adaptive immune response, from thedata available from hemophilia clinical trials. Thus, there is a need todevelop novel strategies which will allow lower doses of vectors to beused to achieve sustained phenotypic correction and limit vector relatedimmune-toxicities.

This Example shows that surface-exposed lysine residues of the VP3region of the VVA2 capsid protein are direct targets for host ubiquitinligases, and mutagenesis of these lysine residues improves transductionefficiency of the AAV vectors.

In silico analysis using a ubiquitination prediction software (UbPred)identified seven lysine residues (K39, K137, K143, K161, K490, K527 andK532) of the wild-type AAV2 capsid could be ubiquitinated. Lysine toArginine mutations in AAV2 Rep/Cap coding plasmid was carried out andhighly purified stocks of a recombinant self-complementary AAV2 vectorsexpressing EGFP [scAAV-CBa-EGFP] were generated in each of the sevenlysine mutant plasmids. The physical particle titres of lysine mutantvectors was comparable to wild-type (WT) scAAV vectors (˜0.5-1×10¹²vgs/mL), suggesting that these mutations did not affect the structure orpackaging ability of mutant capsids.

scAAV vectors containing WT or each of the seven lysine mutant capsidswere then evaluated for their transduction potential in vitro.Approximately 8×10⁴ HeLa or HEK293 cells were mock-infected or infectedwith AAV at different multiplicities of infection (MOI) including 500,2000 or 5000 vgs/cell. Forty-eight hours post-infection, transgene(EGFP) expression was measured by fluorescence microscopy and byflow-cytometry.

The results presented in FIG. 15) demonstrated that the K532R mutantvector significantly increased gene expression in both HeLa (18×) andHEK 293 (9×) cells in vitro, when compared to the WT-AAV2 vector. Theincreased transduction efficacy of the K532R vector was consistentacross three different MOIs tested, with an average increase of 10-foldover the WT vector.

Example 9—AAV Vector-Mediated Activation of Canonical and AlternativeNF-KB Pathways In Vivo

Infection of HeLa cells with adeno-associated viral (AAV) vectors invitro results in activation of the alternative pathway of NF-KB, acentral regulator of cellular immune and inflammatory responses. Inaddition, activation of the alternative, but not the canonical pathway,regulates AAV-mediated transgene expression in these cells.

This example defines a role for NF-KB in liver-directed AAV-mediatedgene transfer in mice. In vivo, AAV-mediated gene transfer results inconsecutive activation of the canonial and the alternative NF-KBpathways. These pathways are thought to drive primarily inflammation(canonical) or adaptive responses (alternative pathway). AAV2 vectorswith the wild-type (WT) or the tyrosine triple-mutant (TM) capsidsactivated the canonical NF-KB pathway within 2 hrs, resulting inexpression of pro-inflammatory cytokines and chemokines (FIG. 14A). Thistransient process is Toll-like receptor 9 (TLR9)-dependent and likelyreflects the initial sensing of the vector genome by antigen-presentingcells. Western blot analyses (FIG. 14B) of liver homogenates prepared 9hrs post-vector delivery, showed abundance of the nuclear p52 proteincomponent of the alternative NF-KB pathway, likely resulting from genetransfer to hepatocytes.

Administration of the NF-KB inhibitor Bay11 prior to gene transfereffectively blocked activation of both pathways. This preventedpro-inflammatory innate immune responses and also dampened anti-AAVcapsid antibody formation (FIG. 14C). Importantly, Bay11 did notinterfere with long-term transgene expression mediated by both the WTand the TM AAV2 vectors (FIG. 14D). These results demonstrated thattransient immuno-suppression with NF-KB inhibitor prior to vectoradministration eliminated inflammation (caused by innate responses), andalso limited adaptive responses.

Example 10—Development of Optimized rAAV3 Vectors: Mechanism ofHigh-Efficiency Transduction of Human Liver Cancer Cells

Of the 10 commonly used AAV serotypes, AAV3 has been reported totransduce cells and tissues poorly. However, the present inventorsdiscovered that AAV3 vectors transduce established human hepatoblastoma(HB) and human hepatocellular carcinoma (HCC) cell lines as well asprimary human hepatocytes extremely efficiently. AAV3 utilizes humanHGFR as a cellular receptor/co-receptor for viral entry.

This Example shows that both extracellular as well as intracellularkinase domains of hHGFR are involved in AAV3 vector entry andAAV3-mediated transgene expression. The results show that (i) AAV3vector-mediated transduction is significantly increased in T47D cells, ahuman breast cancer cell line that expresses undetectable levels of theendogenous hHGFR, following stable transfection and over-expression ofhHGFR (FIG. 15A); (ii) the tyrosine kinase activity associated withhHGFR negatively affects the transduction efficiency of AAV3 vectors(FIG. 15B, FIG. 15C); (iii) the use of proteasome inhibitorssignificantly improves AAV3 vector-mediated transduction; (iv)site-directed mutagenesis of specific surface-exposed tyrosine residueson the AAV3 capsid leads to improved transduction efficiency; and (v) aspecific combination of two tyrosine-mutations further improves theextent of transgene expression (FIG. 15D). These AAV3 vectors can beuseful for the gene therapy of liver cancer in humans.

Example 11—Site-Directed Mutagenesis of Surface-Exposed Lysine ResiduesLeads to Improved Transduction by rAAV2 and rAAV8 Vectors in MurineHepatocytes In Vivo

The ubiquitin-proteasome pathway plays a critical role in theintracellular trafficking of recombinant AAV2 vectors, which negativelyimpacts the transduction efficiency of these vectors. The primary signalfor ubiquitination is phosphorylation of specific surface-exposedtyrosine (Y), serine (S), and threonine (T) residues on the AAV2capsids; the removal of some of these residues significantly increasesthe transduction efficiency of the wild-type (WT) AAV2 vectors.

This Example illustrates that site-directed mutagenesis ofsurface-exposed lysine residues prevented ubiquitination of AAV2capsids, which in turn, prevented vector degradation by cellularproteasomal machinery, thereby producing improved vectors for deliveringtherapeutic or diagnostic polynucleotides to selected mammalian cells.

AAV2 vectors with a single mutation in the surface-exposed lysine (K)residues (K258, K490, K527, K532, K544, 549, and K556) with glutamicacid (E) were prepared and analyzed. The transduction efficiency ofK490E, K544E, K549E, and K556E scAAV2 vectors expressing the EGFPreporter gene increased as much as 5-fold, when compared with thecorresponding unmodified WT AAV2 vectors (see FIG. 16). Of the exemplaryconstructs analyzed in this study, the K556E single mutant had thehighest transduction efficiency (with a transduction rate of 2,000vgs/cell in vitro in Hela cells) among the lysine-substituted mutants.Similar results were also obtained when 1×10¹⁰ vgs of each vector wasdelivered intravenously to C57BL/6 mice in vivo, and the transgeneexpression in hepatocytes evaluated at 2-weeks' post-injection.Bioluminescence imaging two weeks post injection following intravenousdelivery of 1×10¹⁰ vgs/animal of either the WT or the lysine-mutatedssAAV2 vectors expressing the firefly luciferase (Fluc) reporter genefurther corroborated these results.

Importantly, two of the most efficient single amino acid residuemutations were combined to generate a double-mutant (K544E+K556E). Thetransduction efficiency of this double-mutant ssAAV2-Fluc vector inmurine hepatocytes in vivo increased by ˜2-fold compared to either ofthe single mutants, and ˜10-fold as compared to the WT, unmodifiedssAAV2 control vector.

AAV8 vectors have previously been shown to transduce murine hepatocytesexceedingly well. As some of the surface-exposed K residues are alsoconserved in this serotype, ssAAV8-Fluc vectors with K530E-, K547E-, orK569E-mutant were also generated. The transduction efficiency of K547Eand K569E ssAAV8-Fluc vectors in murine hepatocytes in vivo increased by˜3-fold and ˜2-fold, respectively, when compared with WT ssAAV8 vectors(FIG. 17A, FIG. 17B, FIG. 17C, FIG. 17D, FIG. 17E, and FIG. 18).

The results (summarized herein in FIG. 19A, FIG. 19B, FIG. 20A and FIG.20B, FIG. 21A-FIG. 21I, FIG. 22A-FIG. 22I, FIG. 23A-FIG. 23F, FIG. 24,and FIG. 25) demonstrated that targeting the surface-exposed lysineresidues could also be exploited to create new, improved AAV-based viralvectors for increased transduction of human cells, and, importantly, newtargeting vectors useful in gene therapy.

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It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims.

All references, including publications, patent applications and patents,cited herein are hereby incorporated by reference to the same extent asif each reference was individually and specifically indicated to beincorporated by reference and was set forth in its entirety herein.

The terms “a” and “an” and “the” and similar referents as used in thecontext of describing the invention are to be construed to cover boththe singular and the plural, unless otherwise indicated herein orclearly contradicted by context.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext.

The use of any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise indicated. No language in the specification should beconstrued as indicating any element is essential to the practice of theinvention unless as much is explicitly stated.

The description herein of any aspect or embodiment of the inventionusing terms such as “comprising”, “having”, “including” or “containing”with reference to an element or elements is intended to provide supportfor a similar aspect or embodiment of the invention that “consists of”,“consists essentially of”, or “substantially comprises” that particularelement or elements, unless otherwise stated or clearly contradicted bycontext (e.g., a composition described herein as comprising a particularelement should be understood as also describing a composition consistingof that element, unless otherwise stated or clearly contradicted bycontext).

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

1. A method for treating a disease or condition, comprisingadministering a therapeutically effective amount of an rAAV particlecomprising an AAV VP3 protein, comprising: (a) a non-lysine amino acidresidue at a position that corresponds to K258, K321, K459, K490, K507,K527, K572, K532, K544, K549, K556, K649, K655, K665, or K706 of thewild-type AAV2 capsid protein of SEQ ID NO:2; (b) a non-lysine aminoacid residue at a position that corresponds to K530, K547, or K569 ofthe wild-type AAV8 capsid protein of SEQ ID NO:8; (c) a non-serine aminoacid residue at a position that corresponds to S261, S264, S267, S276,S384, S458, S468, S492, S498, S578, S658, S662, S668, S707, or S721 ofthe wild-type AAV2 capsid protein of SEQ ID NO:2; (d) a non-threonineamino acid residue at a position that corresponds to T251, T329, T330,T454, T455, T503, T550, T592, T581, T597, T491, T671, T659, T660, T701,T713, or T716 of the wild-type AAV2 capsid protein of SEQ ID NO:2;and/or (e) a non-tyrosine amino acid residue at a position thatcorresponds to Y252, Y272, Y444, Y500, Y700, Y704, or Y730 of thewild-type AAV2 capsid protein of SEQ ID NO:2.
 2. The method according toclaim 1, wherein the AAV VP3 protein comprises a non-lysine amino acidresidue at a position that corresponds to K459, K490, K532, K544, orK556 of the wild-type AAV2 capsid protein of SEQ ID NO:2.
 3. The methodof claim 1, wherein the AAV VP3 protein comprises a non-lysine aminoacid residue at a position that corresponds to K530, K547, or K569 ofthe wild-type AAV8 capsid protein of SEQ ID NO:8.
 4. The method of claim1, wherein the AAV VP3 protein comprises non-lysine amino acid residuesat positions correspond to K544 and K566 of the wild-type AAV2 capsidprotein of SEQ ID NO:2.
 5. The method of claim 1, wherein the non-lysineamino acid residue of the AAV VP3 protein is selected from glutamic acid(E), arginine (R), serine (S), or isoleucine (I).
 6. The method of claim1, wherein the AAV VP3 protein comprises a glutamic acid (E) amino acidresidue at a position that corresponds to K258, K321, K459, K490, K507,K527, K572, K532, K544, K549, K556, K649, K655, or K706 of the wild-typeAAV2 capsid protein of SEQ ID NO:2.
 7. The method of claim 1, whereinthe AAV VP3 protein comprises glutamic acid (E) amino acid residues atpositions correspond to K544 and K566 of the wild-type AAV2 capsidprotein of SEQ ID NO:2.
 8. The method of claim 1, wherein the AAV VP3protein comprise a glutamic acid (E) amino acid residue at a positionthat corresponds to K530, K547, or K569 of the wild-type AAV8 capsidprotein of SEQ ID NO:8.
 9. The method of claim 1, wherein the AAV VP3protein comprises a non-serine amino acid residue at a position thatcorresponds to 5662 of the wild-type AAV2 capsid protein of SEQ ID NO:2.10. The method of claim 1, wherein the non-serine amino acid residue ofthe AAV VP3 protein is selected from valine (V), aspartic acid (D), orhistidine (H).
 11. The method of claim 1, wherein the AAV VP3 proteincomprises a valine residue at a position that corresponds to S662 of thewild-type AAV2 capsid protein of SEQ ID NO:2. 12-17. (canceled)
 18. Themethod of claim 1, wherein the disease or condition is cancer, diabetes,autoimmune disease, kidney disease, cardiovascular disease, pancreaticdisease, intestinal disease, liver disease, neurological disease,neuromuscular disorder, neuromotor deficit, neuroskeletal impairment,neurological disability, neurosensory dysfunction, stroke, ischemia,eating disorder, α₁-antitrypsin (AAT) deficiency, Batten's disease,Alzheimer's disease, Huntington's disease, Parkinson's disease, skeletaldisease, trauma, or pulmonary disease in a mammal.
 19. (canceled) 20.The method of claim 1, wherein the step of administering, comprisestransducing a population of mammalian cells with a composition thatcomprises an effective amount of the rAAV particle comprising the AAVVP3 protein.
 21. The method according to claim 20, wherein the rAAVparticle comprises a VP3 protein comprising a non-lysine amino acidresidue at a position that corresponds to K459, K490, K532, K544, orK556 of the wild-type AAV2 VP3 protein of SEQ ID NO:2 or K530, K547, orK569 of the wild-type AAV8 capsid protein of SEQ ID NO:8.
 22. The methodaccording to claim 21, wherein the non-lysine amino acid residue isglutamic acid (E), arginine (R), serine (S), or isoleucine (I).
 23. Themethod according to claim 21, wherein the non-lysine amino acid residueis glutamic acid (E).
 24. (canceled)
 25. The method according to claim20, wherein the mammalian cells are endothelial, epithelial, vascular,liver, lung, heart, pancreas, intestinal, kidney, muscle, bone,dendritic, cardiac, neural, blood, brain, fibroblast, or cancer cells.